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The amino acid position within a histone sequence and the chemical nature of post-translational modifications (PTMs) are essential for elucidating the "Histone Code". Previous work has shown that PTMs induce specific biological responses and are good candidates as biomarkers for diagnostics. Here, we evaluate the analytical advantages of trapped ion mobility (TIMS) with parallel accumulation-serial fragmentation (PASEF) and tandem mass spectrometry (MS/MS) for bottom-up proteomics of model cancer cells. The study also considered the use of nanoliquid chromatography (LC) and traditional methods: LC-TIMS-PASEF-ToF MS/MS vs nLC-TIMS-PASEF-ToF MS/MS vs nLC-MS/MS. The addition of TIMS and PASEF-MS/MS increased the number of detected peptides due to the added separation dimension. All three methods showed high reproducibility and low RSD in the MS domain (<5 ppm). While the LC, nLC and TIMS separations showed small RSD across samples, the accurate mobility (1/K0) measurements (<0.6% RSD) increased the confidence of peptide assignments. Trends were observed in the retention time and mobility concerning the number and type of PTMs (e.g., ac, me1-3) and their corresponding unmodified, propionylated peptide that aided in peptide assignment. Mobility separation permitted the annotation of coeluting structural and positional isomers and compared with nLC-MS/MS showed several advantages due to reduced chemical noise.

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Milk is one of the most widely consumed foods globally. To protect consumer interests, it is essential to establish an analytical method to detect the degree of heating in milk. A novel approach using nano liquid chromatography-orbitrap fusion mass spectrometer was developed for screening and identifing thermally sensitive peptides markers in the milk heating process (below 100 °C). This method integrates untargeted proteomics and chemometric tools to analyze protein quantitation data from differently heat-treated milk. Thirteen potential markers were screened out and identified, and further confirmed using by standard substances. Then, the accurate concentrations of 13 potential markers determined by isotope-dilution ultra-performance liquid chromatography-tandem triple quadrupole mass spectrometry were further mining the highly specific and thermally sensitive peptides markers. And Four peptides-INLFDTPLETQYVR, FELLGCELNGCTEPLGLK, QFQFIQVAGR, and GEADALNLDGGYIYTAGK-were selected as marker peptides to differentiate normal pasteurized milk from overheated pasteurized milk. The concentrations of INLFDTPLETQYVR ranges from 150 ± 11 µg/L to 350 ± 23 µg/L, while the concentrations of FELLGCELNGCTEPLGLK ranges from 40 ± 5 µg/L to 92 ± 3 µg/L, can distinguish normal pasteurized milk from overheated pasteurized milk. QFQFIQVAGR indicates overheated pasteurized milk at 230 ± 21 µg/L, and GEADALNLDGGYIYTAGK signifies 750 ± 43 µg/L. This study provides new insights for distinguishing overheated pasteurized milk.

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Miniaturized weak affinity chromatography is emerging as an interesting alternative to conventional biophysical tools for performing fragment-screening studies in the context of fragment-based drug discovery. In order to push back the analytical limits, it is necessary not only to control non-specific interactions with chromatographic support, but also to adapt this methodology by comparing the results obtained on an affinity column to a control column. The work presented in this study focused on fragment screening that targets a model membrane protein, the adenosine A2A receptor, embedded in nanodiscs (NDs) as biomimetic membranes. By studying the retention behavior of test fragment mixtures on supports modified with different types of NDs, we were able to determine the contribution of ND-related non-specific interactions, in particular the electrostatic effect of anionic phospholipids and the hydrophobic effect of neutral phospholipids. Different strategies for the preparation of control columns (empty NDs, orthosteric site blocking) were investigated and are presented for the first time. With these two types of control columns, the screening enabled the identification of two new fragments of AA2AR, which were confirmed by competition experiments and whose Kd values, estimated directly during the screening or after the competition experiments in frontal mode, were in good agreement.

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Poly(butyl methacrylate-co-ethylene dimethacrylate) monolith was in situ prepared in a liquid chromatography capillary column with a 75 µm internal diameter. This monolith offered high permeability (5.3 ± 10-14 m2) and good peak capacity (140 for a 15 cm column length at 300 nl/min with a 20 min gradient time). This is exemplified by its separation ability in reversed mode for subunit analysis of monoclonal antibodies after IdeS digestion (middle-up analysis). The potential of this column was also illustrated for the fast analytical control of therapeutic monoclonal antibodies in standardized infusion bags prepared in advance in a pharmacy department. Linearity analysis revealed the column's capability for accurate quantification analysis of the different dose bandings (in mg) of monoclonal antibodies in <2 min. In addition, lifetime analysis data indicated that the column can be highly reproducible and has a long lifetime with stable and low back pressure. The variations observed on the peak shape and area between unstressed (intact) and stressed monoclonal antibodies indicated that our nano liquid chromatographic method was stability indicating. In addition, using a gradient elution mode, the presence of minor components in the infusion bags was visualized.

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Recombinant human follistatin (rhFST) is a potential performance-enhancing agent owing to its stimulating effect on muscle growth. Administration of rhFST to athletes is prohibited in human sports by the World Anti-Doping Agency (WADA) and in horseracing according to Article 6 of the International Agreement on Breeding, Racing and Wagering published by the International Federation of Horseracing Authorities (IFHA). For effective control of the potential misuse of rhFST in flat racing, methods for screening and confirmatory analysis are required. This paper describes the development and validation of a complete solution for detecting rhFST and confirming its presence in plasma samples collected from racehorses. A high-throughput analysis of rhFST with a commercially available enzyme-linked immunosorbent assay (ELISA) was evaluated for the screening of equine plasma samples. Any suspicious finding would then be subjected to a confirmatory analysis using immunocapture, followed by nano-liquid chromatography/high-resolution tandem mass spectrometry (nanoLC-MS/HRMS). The confirmation of rhFST by nanoLC-MS/HRMS was achieved by comparing the retention times and relative abundances of three characteristic product-ions with those from the reference standard in accordance with the industry criteria published by the Association of Official Racing Chemists. The two methods achieved comparable limit of detection (~2.5-5 ng/mL) and limit of confirmation (2.5 ng/mL or below), as well as adequate specificity, precision and reproducibility. To our knowledge, this is the first report of the screening and confirmation methods for rhFST in equine samples.

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Here, we report the preparation and application of two new chiral monoliths for the enantioseparation of chiral drugs in nano-LC. Using 3­chloro-2-hydroxypropylmethacrylate (HPMA-Cl, 2) as a precursor monomer, two different chiral monomers namely, Nα-Boc-Lys-HPMA (3A) and Nα-Fmoc-Lys-HPMA (3B) were synthesized and used for the preparation of chiral polymer monoliths. The first monolithic column (referred to as monolith I) was prepared by an in-situ polymerization of Nα-Boc-Lys-HPMA as the chiral monomer and ethylene dimethacrylate while the second monolithic column (referred to as monolith II) was prepared by an in-situ polymerization of Nα-Fmoc-Lys-HPMA as the chiral monomer and ethylene dimethacrylate as the crosslinker. Methanol and 1-propanol were used as the porogenic solvents. The prepared chiral monoliths were investigated for the enantioseparation of chiral drugs, including ß-blockers (e.g., atenolol, propranolol, metoprolol) and anti-inflammatory drugs (e.g., ketoprofen, ibuprofen, flurbiprofen, naproxen, etodolac). The enantioseparation could be achieved via the formation of π-π interactions on the aromate-rich and aromate-poor chiral molecules while enantioseparation mechanism of chiral drugs included mostly π-π interactions and hydrogen bonding. Monolith II showed better enantioselectivity than Monolith I and the resolution values up to 2.12 were successfully achieved.

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Since the late 1990s, cathepsin K cleavage sites in type I collagen have been extensively studied due to its ability to release bone resorption biomarkers such as CTX and NTX. However, gel-based methods and N-sequencing used in these studies lack sensitivity, especially for small to medium peptides. In this work, we propose a degradomics mass spectrometry-based workflow that combines protein digestion, Nano-LC-UDMSE, and several software tools to identify cathepsin K cleavage sites. This workflow not only identified previously known cleavage sites, but also discovered new ones. Multiple cleavage hotspots were found and described in type I α1 and type I α2 collagen, many of which coincided with pyridinoline crosslinks, known to stabilize the triple helix. Our results allowed us to establish a chronology of digestion and conclude that cathepsin K preferentially cleaves the extremities of type I collagen before the helical part. We also found that cathepsin K preferentially cleaves amino acid residues with long and hydrophobic lateral chains at the beginning of digestion, whereas no preferred amino acid residues were identified later in the digestion. In conclusion, our workflow successfully identified new cleavage sites and can be easily applied to other proteins or proteases.

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This work presents a study on the application of wall open tubular column (WCOT) in liquid chromatography coupled with tandem mass spectrometry. Each process step reports the column preparation method in detail, subdivided into column pretreatment, silanization, stationary phase coating, and immobilization. Then, an evaluation of the parameters that can affect the efficiency of these columns was made. Atrazine, clomazone, and metolachlor were used as probes during this step. Factors such as stationary phase composition, length, internal diameter, stationary phase mass employed, and injection volume were investigated. In addition, with the help of Knox and Poppe graphs, the columns' performance was evaluated to determine the optimal flow rate and the speed-efficiency relationship, respectively. Based on the results, the best configurations for the WCOT column application to the LC system were defined: length-8 m; inner diameter-25 µm; mass of OV-210-2.5% m/v; and, injection volume-100 nL. Finally, the optimized WCOT column developed in this work was coupled with a commercially-packed trapping column in the nano liquid chromatography system (nanoLC). In this configuration, more significant results were obtained regarding separation resolution, with Rs = 5.9 achieved for the most retained pair of analytes (clomazone and metolachlor).

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Although LC-MS with atmospheric pressure ionization (API) sources is the primary technique used in modern bioanalytical studies, electron ionization mass spectrometry (EI-MS) can provide some substantial advantages over it. EI-MS is a matrix effect-free technique that provides reproducible and comparable mass spectra, serving as a compound fingerprint for easy identification through automated comparison with spectral libraries. Leveraging EI-MS in biochemical studies can yield critical analytical benefits for targeted and untargeted analyses. However, to fully utilize EI-MS for heavy and non-volatile molecules, a new technology that enables the coupling of liquid chromatography with EI-MS is needed. Recent advancements in nanoLC have addressed the compatibility issues between LC and EI-MS, and innovative interfacing strategies such as Direct-EI, liquid electron ionization (LEI), and Cold-EI have extended the application of EI-MS beyond the determination of volatile organic molecules. This review provides an overview of the latest developments in nanoLC-EI-MS interfacing technologies, discussing their scope and limitations. Additionally, selected examples of nanoLC-EI-MS applications in the field of biochemical analysis are presented, highlighting the potential prospects and benefits that the establishment of this technique can bring to this field.

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A new capillary monolithic stationary phase was synthesized for the purification of histidine tagged proteins by immobilized metal affinity micro-chromatography (µ-IMAC). For this purpose, mercaptosuccinic acid (MSA) linked-polyhedral oligomeric silsesquioxane [MSA@poly(POSS-MA)] monolith 300 µm in diameter was obtained by thiol-methacrylate polymerization using methacryl substituted-polyhedral oligomeric silsesquioxane (POSS-MA) and MSA as the thiol functionalized agent in a fused silica capillary tubing. Ni(II) cations were immobilized onto the porous monolith via metal-chelate complex formation with double carboxyl functionality of bound MSA segments. µ-IMAC separations aiming the purification of histidine tagged-green fluorescent protein (His-GFP) from Escherichia coli extract were carried out on Ni(II)@MSA functionalized-poly(POSS-MA) [Ni(II)@MSA@poly(POSS-MA)] capillary monolith. His-GFP was succesfully isolated by µ-IMAC on Ni(II)@MSA@poly(POSS-MA) capillary monolith with the isolation yield of 85 % and the purity of 92 % from E. coli extract. Higher His-GFP isolation yields were obtained with lower His-GFP feed concentrations and lower feed flow rates. The monolith was used for consecutive His-GFP purifications with a tolerable decrease in equilibrium His-GFP adsorption over five runs.

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Enantiomers show different behaviors in interaction with the chiral environment. Due to their identical chemical structure and their wide application in various industries, such as agriculture, medicine, pesticide, food, and so forth, their separation is of great importance. Today, the term "nano" is frequently encountered in all fields. Technology and measuring devices are moving towards miniaturization, and the usage of nanomaterials in all sectors is expanding substantially. Given that scientists have recently attempted to apply miniaturized techniques known as nano-liquid chromatography/capillary-liquid chromatography, which were originally accomplished in 1988, as well as the widespread usage of nanomaterials for chiral resolution (back in 1989), this comprehensive study was developed. Searching the terms "nano" and "enantiomer separation" on scientific websites such as Scopus, Google Scholar, and Web of Science yields articles that either use miniaturized instruments or apply nanomaterials as chiral selectors with a variety of chemical and electrochemical detection techniques, which are discussed in this article.

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DNA methylation is one of the epigenetic modifications at the 5-carbon of cytosine to form 5-methyl-2'-deoxycytidine (5mdC). In mammalian cells, 5mdC can be transferred to 5-hydroxymethyl-2'-deoxycytidine (5hmdC) by ten-eleven translocation (TET), and 5hmdC is further oxidized to 5-formyl-2'-deoxycytidine (5fodC) and 5-carboxyl-2'-deoxycytidine (5cadC). In the present work, we developed a highly sensitive nano liquid chromatographic method for the determination of 5mC and 5hmC with zwitterionic monolithic capillary column. The conditions for the preparation of zwitterionic monolithic capillary column were systematically optimized. The monolithic capillary column displayed high column efficiency for nucleoside dA (190,000 plates/m) and allowed the baseline separation of 10 standard nucleosides in HILIC mode. The detection sensitivity was improved significantly by using the large volume injection combined with sample stacking onto the head of the column when sample was dissolved in high content organic solvent (ACN: H2O = 97:3). The limit of detection for 5mdC and 5hmdC were determined as 4 nM and 3 nM, respectively, and the corresponding limit of quantification were determined as 12 nM and 10 nM, respectively. Further, the zwitterionic monolithic capillary column can be easily utilized in nano-LC and mass spectrometry coupling for qualitative analysis of 5mdC, 5hmdC, 5fodC and 5cadC in real mouse brain sample. The whole genomic DNA methylation levels in mouse brain sample can be easily determined with UV detection.

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A method for the preparation of poly(N-vinylpyrrolidone-co-pentaerythritol triacrylate copolymerization)-based monolithic capillary column was reported for the separation of polar small molecular weight compounds with nano-liquid chromatography in hydrophilic interaction chromatography mode. The monolithic columns were prepared by in situ copolymerization of N-vinylpyrrolidone and a cross-linker pentaerythritol triacrylate in a binary porogenic agent consisting of methanol and water. The composition of the polymerization solution was systematically optimized in terms of column permeability, theoretical plate number, asymmetric factor, and retention factor. A typical hydrophilic chromatography retention mechanism was observed with a mobile phase composed of a high content of organic solvent. The preparation method is simple and robust, the precursor N-vinylpyrrolidone is chemically stable, cheap, and easily available. The N-vinylpyrrolidone-based hydrophilic interaction chromatography stationary phase displays satisfactory separation selectivity for a range of polar test analytes, including benzoic acid derivatives, nucleosides, and phenols.

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In recent years, a trend toward instrument miniaturization has led to the development of new and sophisticated analytical systems, such as nano-liquid chromatography (nano-LC), which has enabled improvements of sensitivity, as well as chromatographic resolution. The growing interest in nano-LC methodology has resulted in a variety of innovative and promising applications. In this article, we review the applications of nano-LC separation methods coupled with mass spectrometry in the analysis of food and environmental samples. An assessment of sample preparation methods and analytical performance are provided, along with comparison to other, more established analytical techniques. Three main groups of compounds that are crucial for food safety assessment are considered in this review: pharmaceuticals (including antibiotics), pesticides, and mycotoxins. Recent practical applications of the nano-LC method in the determination of these compounds are discussed. Furthermore, we also focus on methods for the determination of various environmental contaminants using nano-LC methods. Future perspectives for the development of nano-LC methods are discussed.

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Advanced quantitative bioanalytical approaches in combination with network analyses allow us to answer complex biological questions, such as the description of changes in protein profiles under disease conditions or upon treatment with drugs. In the present work, three quantitative proteomic approaches-either based on labelling or not-in combination with network analyses were applied to a new in vitro cellular model of nonalcoholic fatty liver disease (NAFLD) for the first time. This disease is characterized by the accumulation of lipids, inflammation, fibrosis, and insulin resistance. Hepatic G2 cells were used as model, and NAFLD was induced by a complex of oleic acid and bovine albumin. The development of the disease was verified by lipid vesicle staining and by the increase in the expression of perilipin-2-a protein constitutively present in the vesicles during NAFLD. The nLC-MS/MS analyses of peptide samples obtained from three different proteomic approaches resulted in accurate and reproducible quantitative data of protein fold-change expressed in NAFLD versus control cells. The differentially regulated proteins were used to evaluate the involved and statistically enriched pathways. Network analyses highlighted several functional and disease modules affected by NAFLD, such as inflammation, oxidative stress defense, cell proliferation, and ferroptosis. Each quantitative approach allowed the identification of similar modulated pathways. The combination of the three approaches improved the power of statistical network analyses by increasing the number of involved proteins and their fold-change. In conclusion, the application of advanced bioanalytical approaches in combination with pathway analyses allows the in-depth and accurate description of the protein profile of an in vitro cellular model of NAFLD by using high-resolution quantitative mass spectrometry data. This model could be extremely useful in the discovery of new drugs to modulate the equilibrium NAFLD health state.

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A 25 µm i.d x 1.2 m length PS-DVB porous layer open tubular column (PLOT) was prepared and assessed in the configuration of a nano liquid chromatography coupled to an electron ionization mass spectrometry system (OT-nanoLC-EI-Ms), via the direct insertion of the column outlet into the ionization source. The developed system's operational parameters were comprehensively studied, and the setup performance was investigated employing both unidimensional and column switching configurations. As a result, the OT-nanoLC-EI-MS system demonstrated competitive applicability in separating non-amenable ESI compounds, such as polyaromatic hydrocarbons (PAHs) and non-amenable GC compounds such as thermolabile pesticides. Furthermore, with excellent chromatographic performance, the PLOT columns can work under more compatible EI-detection conditions - such as the elution with 100% organic solvent. For example, PAHs retention factors ranged between 1.5 and 2.2 for 100% MeCN mobile phase, and more than 33,000 plates per meter for naphthalene at 50 nL/min flow rate. In analyzing thermolabile pesticides, the column switching PLOT-nanoLC-EI-MS system provided LODs of 25 µg/L, demonstrating suitable intra e interday reproducibility (% RSD < 13%, n = 3), and possibilities the direct injection of raw samples with suitable robustness.

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In this paper enantiomers of selected chiral agrochemicals representing various structural classes were separated by using nano-liquid chromatography (nano-LC) and capillary electrochromatography (CEC) employing a capillary column packed with silica particles containing immobilized amylose tris(3­chloro-5-methylphenylcarbamate) (i-ADMPC) as a chiral selector (CS). Special attention was paid to peak dispersion in nano-LC and CEC instruments used in order to make comparison between these two techniques more reliable. Enantioseparations were studied utilizing methanol (MeOH) or acetonitrile-water (ACNH2O), both containing 5 mM of ammonium acetate as the mobile phases (MPs). The tested chiral stationary phase (CSP), containing 20% (w/w) of the neutral CS onto native silica, allowed the generation of sufficiently strong electroosmotic flow (EOF) to observe separation of enantiomers of studied agrochemicals in a reasonable time also in CEC mode. Modestly higher efficiencies and enantioresolutions were obtained in CEC than in nano-LC. Just a moderate preference of CEC over nano-LC in this particular study can be explained with a significant mass transfer resistance through the CSP that is caused due to high content of the CS in CSP.

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Chloramphenicol (CAP) is an effective antibiotic with broad spectrum against gram-positive and gram-negative bacteria, while it is used to treat various infections in animals. Although CAP is banned for usage in the livestock products including, milk, honey, seafood, and royal jelly, CAP is still often detected in foods of animal origin, posing a threat to consumer health. The use of CAP is restricted in many countries due to its side effect in human metabolic process according to the Expert Committee on Food Additives (ECFA) recommendation. Chloramphenicol glucuronide (CAPG) is also a metabolic product of CAP, which may be a hazardous chemical for human health. Therefore, the development of sensitive separation and quantification method is an important issue, especially for food safety. Herein, we reported the preparation and application of a monolithic nano-column for CAP and CAPG analyses in foods by ProFlow Nano liquid chromatography (ProFlow Nano LC). The monolithic nano-column was prepared by an in situ polymerization using 3-chloro-2-hydroxypropylmethacrylate (HPMA-Cl) and ethylene dimethacrylate (EDMA) and followed graphene oxide (GO) modification. After characterization, the monolithic nano-column was used for the analysis of CAP and CAPG in honey and milk samples by ProFlow Nano LC. The whole method was validated in terms of linearity, sensitivity, precision, recovery, and repeatability, while it led to obtain high sensitivity with limit of quantification was found as 0.02 µg/kg for CAP. Limit of quantification for CAPG was found as 0.08 µg/kg. The developed method with monolithic nano-column was optimized to achieve very sensitive analyses of CAP and CAPG in the food samples. The applicability of the nano-column was successfully demonstrated by the analysis of CAP and CAPG in milk and honey samples. PRACTICAL APPLICATION: This article describes the preparation and application of a monolithic nano-column for the separation and determination of chloramphenicol and chloramphenicol glucuronide in food samples by ProFlow Nano LC. The use of new and advanced techniques is a crucial issue in the food science and technology. In this sense, this study demonstrated a new food analysis method using advanced instrumental technique with a homemade monolithic nano-column.

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This article summarizes our cooperation with the research group of Prof. Yoshio Okamoto at Nagoya University during the period of time between 1992 and 2005. Although the text deals entirely with enantioseparations in high-performance liquid chromatography, capillary electrophoresis, and capillary electrochromatography, this is not a detailed review in any of these areas. The text highlights selected aspects of these techniques, which have been the subject of our joint research and in part their reflection in follow-up research by our and other research groups. Together with more systematically studied topics, aspects such as ultrafast separation of enantiomers, uncommonly high separation factor of enantiomers and other related issues are also addressed.

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In this study three miniaturized liquid chromatography (LC) instruments have been evaluated and compared for the analysis of caffeine in dietary supplements, namely a benchtop capillary LC (capLC) system, a benchtop nano LC (nanoLC)system and a portable LC system. Commercial products derived from different sources of caffeine have been analyzed. Under optimized conditions, the methods based on benchtop systems were superior in terms of sensitivity. The limits of detection (LODs) found with the capLC and nanoLC systems were 0.01 and 0.003 µg mL-1, respectively, whereas the LOD obtained with the portable LC instrument was of 1 µg mL-1. The portable LC-based method was superior in terms of simplicity and throughput (total analysis time < 15 min). On the basis of the results obtained, a new method for the rapid measurement of caffeine in dietary supplements by portable miniaturized LC is presented. This method provided good linearity within the 1-20 µg mL-1 interval, and it allowed the quantification of caffeine even in products derived from decaffeinated green coffee extracts. The contents of caffeine found with the proposed portable LC method in the real samples analyzed ranged from 1.38 to 7 mg per gram of product, which were values statistically equivalent to those found with the benchtop capLC and nanoLC methods, being the precision, expressed as relative standard deviation (RDS), of 2 -14% (n = 3). The proposed portable LC based method can be used as a simple and rapid alternative to estimate the quality, effectiveness and safety of dietary supplements, regarding their caffeine content.

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