Sensitive Detection and Quantification of Oxygenated Compounds in Complex Samples Using GC-Combustion-MS.

This work introduces a new element-selective gas chromatography detector for the accurate quantification of traces of volatile oxygen-containing compounds in complex samples without the need for specific standards. The key to this approach is the use of oxygen highly enriched in 18O as the oxidizing gas in a combustion unit (800 °C) that allows us to directly and unambiguously detect the natural oxygen present in the GC-separated compounds through its incorporation into the volatile species formed after their combustion and their subsequent degradation to 16O in the ion source. The unspecific signal due to the low 16O abundance in the oxidizing gas could be compensated by measuring the m/z 12 that comes as well from the CO2 degradation. Equimolarity was proved with several O-containing compounds with different sizes and functionalities. A detection limit of 28 pg of injected O was achieved, which is the lowest ever reported for any GC detector, which barely worsened to 55 and 214 pg of O when the oxygenate partially or completely coeluted with a very abundant matrix compound. Validation was attained by the analysis of a SRM to obtain accurate (99-103%) and precise (1-4% RSD) results. Robustness was tested after spiking a hydrotreated diesel with 10 O-compounds at the ppm level, which could be discriminated from the matrix crowd and quantified (mean recovery of 102 ± 9%) with a single generic standard. Finally, it was also successfully applied to easily spot and quantify the 33 oxygenates naturally present in a complex wood bio-oil sample.

Relative and Transport Efficiency-Independent Approach for the Determination of Nanoparticle Size Using Single-Particle ICP-MS.

Herein, we introduce the first relative single-particle inductively coupled plasma mass spectrometry (spICP-MS) approach where size calibration is carried out using the target NP itself measured under different instrumental conditions without external dependence on the complex and prone-to-error determination of transport efficiency or mass flux calibrations, in contrast to most spICP-MS approaches. The simple approach proposed allows determining gold nanoparticle (AuNP) sizes, with errors ranging from 0.3 to 3.1% (corroborated by HR-TEM). It has been demonstrated that the changes observed in the single-particle histograms obtained for a suspension of AuNPs under different sensitivity conditions (n = 5) are directly and exclusively related to the mass (size) of the target AuNP itself. Interestingly, the relative nature of the approach shows that once the ICP-MS system has been calibrated with a generic NP standard, it is no longer necessary to repeat the calibration for the size determination of different unimetallic NPs carried out along time (at least 8 months), independently of their size (16-73 nm) and even nature (AuNP or AgNP). Additionally, neither the NP surface functionalization with biomolecules nor protein corona formation led to significant changes (relative errors slightly increased 1.3- to 1.5-fold, up to 7%) in the NP size determination, in contrast to conventional spICP-MS approaches where relative errors increased 2- to 8-fold, up to 32%. This feature could be especially valuable for the analysis of NPs in real samples without the need of matrix-matched calibration.

Potential of GC-Combustion-MS as a Powerful and Versatile Nitrogen-Selective Detector in Gas Chromatography.

Here, we show the potential and applicability of the novel GC-combustion-MS approach as a nitrogen-selective GC detector. Operating requirements to achieve reproducible and compound-independent formation of volatile NO species as a selective N-signal during the combustion step are described. Specifically, high temperatures (≥1000 °C) and post-column O2 flows (0.4 mL min-1 of 0.3% O2 in He) turned out to be necessary when using a vertical oven without makeup flow (prototype #1). In contrast, the use of a horizontal oven with 1.7 mL min-1 He as an additional makeup flow (prototype #2) required milder conditions (850 °C and 0.2 mL min-1). A detection limit of 0.02 pg of N injected was achieved, which is by far the lowest ever reported for any GC detector. Equimolarity, linearity, and peak shape were also adequate. Validation of the approach was performed by the analysis of a certified reference material obtaining accurate (2% error) and precise (2% RSD) results. Robustness was tested with the analysis of two complex samples with different matrices (diesel and biomass pyrolysis oil) and N concentration levels. Total N determined after the integration of the whole chromatograms (524 ± 22 and 11,140 ± 330 µg N g-1, respectively) was in good agreement with the reference values (497 ± 10 and 11,000 ± 1200 µg N g-1, respectively). In contrast, GC-NCD results were lower for the diesel sample (394 ± 42 µg N g-1). Quantitative values for the individual and families of N species identified in the real samples by parallel GC-MS and additional GC × GC-MS analyses were also obtained using a single generic internal standard.

Signal amplification strategies for clinical biomarker quantification using elemental mass spectrometry.

The current trends in modern medicine towards early diagnosis, or even prognosis, of different diseases have brought about the need for the corresponding biomarker detection at ever lower levels in really complex matrices. To do so, it is necessary to use proper extremely sensitive detection techniques such as elemental mass spectrometry. However, target labelling with metals for subsequent sensitive ICP-MS detection falls short nowadays even if resorting to inorganic nanoparticles containing a high number of detectable elements. Thus, new amplification strategies are being proposed to face this analytical challenge that will be critically discussed in this paper. Fundamentals of different novel strategies developed to achieve signal amplification and sensitive elemental mass spectrometry detection are here discussed. Some representative examples of relevant clinical applications are highlighted, along with future prospects and challenges.

Combined Molecular and Elemental Mass Spectrometry Approaches for Absolute Quantification of Proteomes: Application to the Venomics Characterization of the Two Species of Desert Black Cobras, Walterinnesia aegyptia and Walterinnesia morgani.

We report a novel hybrid, molecular and elemental mass spectrometry (MS) setup for the absolute quantification of snake venom proteomes shown here for two desert black cobra species within the genus Walterinnesia, Walterinnesia aegyptia and Walterinnesia morgani. The experimental design includes the decomplexation of the venom samples by reverse-phase chromatography independently coupled to four mass spectrometry systems: the combined bottom-up and top-down molecular MS for protein identification and a parallel reverse-phase microbore high-performance liquid chromatograph (RP-µHPLC) on-line to inductively coupled plasma (ICP-MS/MS) elemental mass spectrometry and electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-QToF MS). This allows to continuously record the absolute sulfur concentration throughout the chromatogram and assign it to the parent venom proteins separated in the RP-µHPLC-ESI-QToF parallel run via mass profiling. The results provide a locus-resolved and quantitative insight into the three desert black cobra venom proteome samples. They also validate the units of measure of our snake venomics strategy for the relative quantification of snake venom proteomes as % of total venom peptide bonds as a proxy for the % by weight of the venom toxins/toxin families. In a more general context, our work may pave the way for broader applications of hybrid elemental/molecular MS setups in diverse areas of proteomics.

Assessment of the Potential and Limitations of Elemental Mass Spectrometry in Life Sciences for Absolute Quantification of Biomolecules Using Generic Standards.

Inductively coupled plasma-mass spectrometry (ICP-MS) has been widely used in Life Sciences for the absolute quantification of biomolecules without specific standards, assuming the same response for generic compounds including complex biomolecules. However, contradictory results have been published on this regard. We present the first critical statistical comparison of the ICP-MS response factors obtained for 14 different relevant S-containing biomolecules (three peptides, four proteins, one amino acid, two cofactors, three polyethylene glycol (PEG) derivatives, and sulfate standard), covering a wide range of hydrophobicities and molecular sizes. Two regular flow nebulizers and a total consumption nebulizer (TCN) were tested. ICP-MS response factors were determined though calibration curves, and isotope dilution analysis was used to normalize the results. No statistical differences have been found for low-molecular-weight biocompounds, PEGs, and nonhydrophobic peptides using any of the nebulizers tested. Interestingly, while statistical differences were still found negligible (96-104%) for the proteins and hydrophobic peptide using the TCN, significantly lower response factors (87-40%) were obtained using regular flow nebulizers. Such differential behavior seems to be related mostly to hydrophobicity and partially to the molecular weight. Findings were validated using IDA in intact and digested bovine serum albumin solutions using the TCN (98 and 100%, respectively) and the concentric nebulizer (73 and 97%, respectively). Additionally, in the case of a phosphoprotein, results were corroborated using the P trace in parallel to the S trace used along the manuscript. This work seems to suggest that ICP-MS operated with regular nebulizers can offer absolute quantification using generic standards for most biomolecules except proteins and hydrophobic peptides.

GC-ICP-MS/MS Instrumental Setup for Total and Speciation Sulfur Analysis in Gasolines using Generic Standards.

Quantitative characterization of sulfur-containing petroleum derivatives is mainly limited by the large number of potential targets present and the matrix effects suffered due to the high-carbon-containing matrices. Herein we describe the instrumental modifications required in a commercial GC-ICP-MS/MS instrument, and their corresponding optimization, for turning it into a compound-independent quantitative technique for both total and speciation sulfur analysis in gasolines. Additionally, carbon-derived matrix effects were made negligible for direct and fast total S analysis, making the use of relatively complex isotope-dilution strategies not necessary anymore. An absolute detection limit of 0.3 pg of S was achieved, which is, to the best of our knowledge, more than 1 order of magnitude below the ones reported for other sulfur GC selective detectors. The precision was below 3% RSD. Total analysis was performed by flow-injection analysis through a transfer line and external calibration, whereas speciation analysis was carried out by chromatographic separation and internal standardization. In both cases, simple generic standards were used, which enabled us to get rid of specific S-containing standards, which were sometimes not available or unstable. The proposed method was successfully applied to total and speciation sulfur analysis of a commercial gasoline sample and validated with a certified-reference-material (ERM-EF213) gasoline. The approach has proved to be simple, fast, robust, and convenient for implementation in routine laboratories, as demonstrated by the successive analyses of 50 gasoline samples in 3 h without any instrumental drift.

Anion-Specific Sulfur Isotope Analysis by Liquid Chromatography Coupled to Multicollector ICPMS.

An accurate method has been developed to measure, in a single analytical run, δ34S in sulfite, sulfate and thiosulfate in water samples by liquid chromatography combined with multicollector inductively coupled plasma mass spectrometry (MC-ICPMS). The method is based on the anionic exchange separation of sulfur species prior to their online isotope ratio determination by MC-ICPMS. Mass bias correction was accomplished by a novel approach based on the addition of an internal sulfur-containing standard to the sample. This innovative approach was compared to the sample-standard bracketing procedure. On-column isotopic fractionation was observed and therefore corrected by external calibration. Isotopic ratios were calculated by linear regression slope (LRS), an advantageous method for transient signals, leading to a combined uncertainty of δ34S below 0.25‰ and a reproducibility below 0.5‰ for the injection of 1 µg of S. The method was successfully applied to the measurement of δ34S in synthetic solutions and environmental water samples. Matrix effects leading to δ34S overestimation were observed for sulfate in some samples with high sodium/sulfate mass ratios. The developed analytical procedure simplifies the δ34S analysis of liquid environmental samples since preparation steps are no longer required and allows the analysis of several sulfur-containing species in a single run.

Enhanced Universal Quantification of Biomolecules Using Element MS and Generic Standards: Application to Intact Protein and Phosphoprotein Determination.

Tools that provide absolute quantification of biomolecules, particularly of proteins and their post-translational modifications, without needing suitable specific standards, are urgently demanded nowadays. To this end, we have significantly improved the recently introduced strategy based on CH4 addition to the plasma for absolute quantification of biomolecules using HPLC-ICP-MS. Addition of CO2 has been optimized and finally selected as a safer, more efficient quantitative strategy that is able to provide constant (<6% error) signal response factor for the six elements assayed (S, P, As, Se, Br, I) under compromised conditions. In the particular case of absolute protein quantification, accuracy and precision attainable for S-based absolute determination of intact proteins using internal and external S-generic standards were compared. Potential for real sample analysis was demonstrated by the high-sensitivity analysis of toxins present in snake venoms. Finally, multielemental speciation capabilities of the approach have been also demonstrated through P and S simultaneous analysis in phosphoproteomics. Simultaneous accurate determination of both absolute protein amount and corresponding phosphorylation degree for intact ß-casein, and even impurity traces of κ and α-s1 isoforms present, has been successfully achieved using a simple mixture of inorganic P and S standards. The lowest detection limits (<1 fmol protein) ever published for S- and P-based intact protein quantification with ICP-MS are reported.

Standardization approaches in absolute quantitative proteomics with mass spectrometry.

Mass spectrometry-based approaches have enabled important breakthroughs in quantitative proteomics in the last decades. This development is reflected in the better quantitative assessment of protein levels as well as to understand post-translational modifications and protein complexes and networks. Nowadays, the focus of quantitative proteomics shifted from the relative determination of proteins (ie, differential expression between two or more cellular states) to absolute quantity determination, required for a more-thorough characterization of biological models and comprehension of the proteome dynamism, as well as for the search and validation of novel protein biomarkers. However, the physico-chemical environment of the analyte species affects strongly the ionization efficiency in most mass spectrometry (MS) types, which thereby require the use of specially designed standardization approaches to provide absolute quantifications. Most common of such approaches nowadays include (i) the use of stable isotope-labeled peptide standards, isotopologues to the target proteotypic peptides expected after tryptic digestion of the target protein; (ii) use of stable isotope-labeled protein standards to compensate for sample preparation, sample loss, and proteolysis steps; (iii) isobaric reagents, which after fragmentation in the MS/MS analysis provide a final detectable mass shift, can be used to tag both analyte and standard samples; (iv) label-free approaches in which the absolute quantitative data are not obtained through the use of any kind of labeling, but from computational normalization of the raw data and adequate standards; (v) elemental mass spectrometry-based workflows able to provide directly absolute quantification of peptides/proteins that contain an ICP-detectable element. A critical insight from the Analytical Chemistry perspective of the different standardization approaches and their combinations used so far for absolute quantitative MS-based (molecular and elemental) proteomics is provided in this review.

Isotope Dilution Mass Spectrometry for Highly Precise Determination of Dissolved Inorganic Carbon in Seawater Aiming at Climate Change Studies.

Dissolved inorganic carbon (DIC) is one of the most important parameters to be measured in seawaters for climate change studies. Its quantitative assessment requires analytical methodologies with overall uncertainties around 0.05% RSD for clear evaluation of temporal trends. Herein, two alternative isotope dilution mass spectrometry (IDMS) methodologies (online and species-specific) using an isotope ratio mass spectrometer (IRMS) and two calculation procedures for each methodology have been compared. As a result, a new method for the determination of DIC in seawaters, based on species-specific IDMS with isotope pattern deconvolution calculation, was developed and validated. A 13C-enriched bicarbonate tracer was added to the sample and, after equilibration and acidification, the isotope abundances at CO2 masses 44, 45, and 46 were measured on an IRMS instrument. Notably, early spiking allows correcting for evaporations and/or adsorptions during sample preparation and storage and could be carried out immediately after sampling. Full uncertainty budgets were calculated taking into account all the factors involved in the determination (initial weights, concentration and isotope abundances of standards, and final IRMS measurements). The average DIC value obtained for CRM seawater agreed very well with the certified value. Propagated precision obtained ranged from 0.035 to 0.050% RSD for individual sample triplicates. Reproducibility, assessed by three independent experiments carried out in different working days, was excellent as well (-0.01% and 0.057%, error and full combined uncertainty, respectively). Additionally, the approach proposed improves on established methods by simplicity, higher throughput (15 min per sample), and lower volume requirements (10 mL).

Controlling Ligand Surface Density on Streptavidin-Magnetic Particles by a Simple, Rapid, and Reliable Chemiluminescent Test.

The use of functionalized magnetic particles is increasing because they simplify the analytical process and yield promising results in a wide range of applications. Particularly, streptavidin-coated magnetic beads offer the possibility of rapid and very efficient grafting of biomolecules. Unfortunately, current methods to monitor and compute this grafting process are cumbersome and scarce. We describe herein a simple, rapid, and reliable chemiluminescent assay we have developed to check the grafting rate of functionalized magnetic beads. The power of the assay also relies on its ability to predict the amount of ligands required to obtain a precise grafting rate. In addition, results were correlated with a more general parameter in material functionalization characterization like surface ligand density. Finally, the assay was validated for a wide variety of biotinylated biomolecule sizes, ranging from small molecules (around 200 Da) to antibodies (around 150 kDa). This approach will allow a precise quantification and prediction of the functionalization of magnetic particles that is of enormous importance for quality control in many applications.

Instrumental Setup for Simultaneous Total and Speciation Analysis of Volatile Arsenic Compounds in Gas and Liquefied Gas Samples.

Although analysis of metals and metalloids, such as arsenic, is widely spread in many different fields, their analysis in gas and liquefied gas samples is still a challenge. A new GC-ICP-MS set up has been developed for their simultaneous total and speciation analysis in gas and liquefied gas samples without the need of a preconcentration step. An arsine in nitrogen standard was used for optimization and evaluation of the system. Good linearity and detection limits in the very low ppt level for both total and speciation analyses were found. Liquefied butane pressurized under nitrogen and doped with arsine and a propylene real sample from a cracker plant were analyzed using both external calibration and standard additions methods. The good match between both quantifying approaches demonstrated almost negligible matrix effects, even for the total analysis. Application of the approach to check repartition of volatile elements or species between gas and liquid phases was performed in the real propylene sample. Finally, its potential applicability for the simultaneous total and speciation analysis of other elements, such as Hg, was also proved.

The use of elemental mass spectrometry in phosphoproteomic applications.

Reversible phosphorylation is one of the most important post-translational modifications in mammalian cells. Because this molecular switch is an important mechanism that diversifies and regulates proteins in cellular processes, knowledge about the extent and quantity of phosphorylation is very important to understand the complex cellular interplay. Although phosphoproteomics strategies are applied worldwide, they mainly include only molecular mass spectrometry (like MALDI or ESI)-based experiments. Although identification and relative quantification of phosphopeptides is straightforward with these techniques, absolute quantification is more complex and usually requires for specific isotopically phosphopeptide standards. However, the use of elemental mass spectrometry, and in particular inductively coupled plasma mass spectrometry (ICP-MS), in phosphoproteomics-based experiments, allow one to absolutely quantify phosphopeptides. Here, these phosphoproteomic applications with ICP-MS as elemental detector are reviewed. Pioneering work and recent developments in the field are both described. Additionally, the advantage of the parallel use of molecular and elemental mass spectrometry is stressed.

Capping of Mn-Doped ZnS Quantum Dots with DHLA for Their Stabilization in Aqueous Media: Determination of the Nanoparticle Number Concentration and Surface Ligand Density.

Colloidal Mn2+-doped ZnS quantum dots (QDs) were synthesized, surface modified, and thoroughly characterized using a pool of complementary techniques. Cap exchange of the native l-cysteine coating of the QDs with dihydrolipoic acid (DHLA) ligands is proposed as a strategy to produce nanocrystals with a strong phosphorescent-type emission and improved aqueous stability. Moreover, such a stable DHLA coating can facilitate further bioconjugation of these QDs to biomolecules using established reagents such as cross-linker molecules. First, a structural and morphological characterization of the l-cysteine QD core was performed by resorting to complementary techniques, including X-ray powder diffraction (XRD) and microscopy tools. XRD patterns provided information about the local structure of ions within the nanocrystal structure and the number of metal atoms constituting the core of a QD. The judicious combination of the data obtained from these complementary characterization tools with the analysis of the QDs using inductively coupled plasma-mass spectrometry (ICP-MS) allowed us to assess the number concentration of nanoparticles in an aqueous sample, a key parameter when such materials are going to be used in bioanalytical or toxicological studies. Asymmetric flow field-flow fractionation (AF4) coupled online to ICP-MS detection proved to be an invaluable tool to compute the number of DHLA molecules attached to the surface of a single QD, a key feature that is difficult to estimate in nanoparticles and that critically affects the behavior of nanoparticles when entering the biological media (e.g., cellular uptake, biodistribution, or protein corona formation). This hybrid technique also allowed us to demonstrate that the elemental composition of the nanoparticle core remains unaffected after the ligand exchange process. Finally, the photostability and robustness of the DHLA-capped QDs, critical parameters for bioanalytical applications, were assessed by molecular luminescence spectroscopy.

Elemental Mass Spectrometry for Absolute Intact Protein Quantification without Protein-Specific Standards: Application to Snake Venomics.

Absolute protein quantification methods based on molecular mass spectrometry usually require stable isotope-labeled analogous standards for each target protein or peptide under study, which in turn must be certified using natural standards. In this work, we report a direct and accurate methodology based on capLC-ICP-QQQ and online isotope dilution analysis for the absolute and sensitive quantification of intact proteins. The combination of the postcolumn addition of 34S and a generic S-containing internal standard spiked to the sample provides full compound independent detector response and thus protein quantification without the need for specific standards. Quantitative recoveries, using a chromatographic core-shell C4 column for the various protein species assayed were obtained (96-100%). Thus, the proposed strategy enables the accurate quantification of proteins even if no specific standards are available for them. In addition, to the best of our knowledge, we obtained the lowest detection limits reported in the quantitative analysis of intact proteins by direct measurement of sulfur with ICPMS (358 fmol) and protein (ranging from 7 to 15 fmol depending on the assayed protein). The quantitative results for individual and simple mixtures of model proteins were statistically indistinguishable from the manufacturer's values. Finally, the suitability of the strategy for real sample analysis (including quantitative protein recovery from the column) was illustrated for the individual absolute quantification of the proteins and whole protein content in a venom sample. Parallel capLC-ESI-QTOF analysis was employed to identify the proteins, a prerequisite to translate the mass of quantified S for each chromatographic peak into individual protein mass.

Determination of the ratio of fluorophore/nanoparticle for fluorescence-labelled nanoparticles.

Optical analysis based on fluorescence labeling has been extensively used for the selective tagging of a wide range of biomedical important targets or for sensing purposes. Fluorescent nanoparticles (NPs) offer interesting properties as labels, as they can be also used as active labels that change their properties upon changes in the environment, such as pH- or distance-dependent fluorescence. In case NPs are not intrinsically fluorescent, they can be made fluorescent by attaching fluorophores to their volume and/or surface. Dye-labelled NPs can produce a highly amplified optical signal compared to a single dye molecule, as there are many dye molecule attached to each NP, providing a great improvement in analytical sensitivity. However, an appropriate control to quantify the fluorophore/NP ratio is required to succeed in the preparation of quantitative platforms matching the required application. Here a methodology to determine such parameter, the fluorophore/NP ratio, is presented. The methodology combines data obtained from UV/Vis absorption spectroscopy for determination of the dye concentration and inductively coupled plasma-mass spectrometry (ICP-MS) analysis for determination of the NP concentration. To validate the approach, it has been applied to the analysis of different sets of fluorophore-NP conjugates prepared using diverse fluorescent dyes (i.e. fluorophores with different structures and emissions) and several types of NPs (i.e. PbS QDs, Au NPs and FePt NPs). The fluorophore-NP conjugates hereby were designed to incorporate the dye directly into an amphiphilic polymer coating. The developed methodology allows for quantification of fluorophore-NP coupling, and therefore, opens up the possibility of selecting controlled conjugates.

An in-depth physicochemical investigation of drug-loaded core-shell UiO66 nanoMOFs.

Nanosized UiO66 are among the most studied MOF materials. They have been extensively applied in various areas, such as catalysis, gas absorption, electrochemistry, chemical sensing, and biomedical applications. However, the preparation of stable nano-sized UiO66 for drug delivery applications is challenging because of the high tendency of UiO66 to aggregate during storage. To address this issue, we coated UiO66 with oligomers made of crosslinked cyclodextrins. The coated UiO66 exhibited a good stability upon storage for more than three weeks, even for low quantities of coating materials. The resulting core-shell UiO66 were characterized using a set of complementary methods including microscopies, spectroscopies, X-ray diffraction, and thermogravimetric investigations. Size distribution was assessed by orthogonal methods. Cisplatin was loaded in the core-shell nanoparticles, followed by an in-depth analysis by asymmetric flow field-flow fractionation (AF4) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS). This method combines the extremely high elemental selectivity and ultratrace detection limits of mass spectrometry with the capacity of AF4 to differentiate the diverse populations present in the sample. Free cisplatin and UiO66-associated cisplatin could be well separated by AF4. AF4-ICP-MS/MS analysis provided the exact drug loading, without the need of separating the nanoparticles from their suspension media. These data suggest the potential of AF4-ICP-MS/MS in the optimization of drug delivery systems.

A plasmonic MNAzyme signal amplification strategy for quantification of miRNA-4739 breast cancer biomarker.

A major challenge in the 21st century is the development of point-of-care diagnostic tools capable to detect and quantify disease biomarkers in a straightforward, affordable, sensitive, and specific manner. The remarkable plasmonic properties of gold nanoparticles (AuNPs) have promoted their use for development of simple methodologies for nucleic acid detection in combination with a variety of oligonucleotides amplification techniques. Here, assemblies of AuNPs with Multicomponent Nucleic Acid enzymes (MNAzymes) has been successfully used in the design of a highly sensitive and simple bioassay for rapid spectroscopic detection and quantification of miRNA-4739 in blood samples. The miRNA selected is a doxorubicin chemoresistant biomarker in breast cancer which overexpression promotes the proliferation, progression, and survival of cancer cells. In this work, two alternatives experimental designs, based on use of MNAzymes and AuNPs, have been optimized and applied for sensitive miRNA-4739 quantification: one based on a traditional direct measurement of wavelength shift and a second non-conventional simple approach based on isolation and measurement of free nanoparticles absorbance. Improvement in sensitivity and, higher measurement accuracy and precision were achieved with the second approach. The developed bioassay provides a detection limit as low as 7 pmolL-1 for miRNA-4739 quantification and performed satisfactory selectivity and well practical applicability by analysis of the miRNA-4739 in blood, demonstrating that the proposed strategy is a promising and suitable spectroscopic method for breast cancer diagnosis thought liquid biopsy of circulating tumoral cells.

Liquid chromatography, chemical oxidation, and online carbon isotope dilution mass spectrometry as a universal quantification system for nonvolatile organic compounds.

A procedure for the universal detection and quantification of polar organic compounds separated by liquid chromatography (LC) based on postcolumn carbon isotope dilution mass spectrometry (IDMS) was developed. The eluent from the LC column is mixed online with a continuous flow of (13)C-enriched sodium bicarbonate, and the sodium persulfate oxidation reaction in acidic media is employed to achieve isotope equilibration. All carbon-containing compounds eluting from the column are oxidized to (12)CO(2) and (13)CO(2), respectively, and the carbon dioxide is separated from the aqueous phase using a gas-permeable membrane. The gaseous carbon dioxide is then carried to the mass spectrometer for isotope ratio measurements. Different water-soluble organic compounds were evaluated using a flow injection configuration to assess the efficiency of the oxidation process. Most water-soluble organic compounds tested showed quantitative oxidation. However, chemical structures involving conjugated C═N double bounds and guanidinium-like structures were found to be resistant to the oxidation and were further studied. For this purpose, (13)C(1)-labeled creatine (with the isotopic label in the guanidinium group) was employed as model compound. Specific conditions for the quantitative oxidation of these compounds required lower flow rates and the addition of metallic catalysts. This novel approach was tested as a universal detection and quantification system for LC. A simple standard mixture of four amino acids was separated under 100% aqueous conditions and quantified without the need for specific standards with good accuracy and precision using potassium hydrogen phthalate as internal standard. The main field of application of the developed method is for the purity assessment of organic standards with direct traceability to the International System of Units (SI).