Sample Preparation Steps Research Articles

Improvement of untargeted proteomics workflow for surfaceome profiling and its evaluation through the implementation of quality controls: Application to multiple myeloma

BackgroundComprehensive surfaceome profiling of cancer cells using mass spectrometry (MS)-based technologies is a valuable approach to identify new antigens that could be targeted by immunotherapies. Multiple myeloma (MM) is an incurable hematological malignancy in which patients suffer from multiple relapses associated with drug resistance. Nevertheless, only three MM-specific antigens are currently targeted by approved immunotherapies which restrain the availability of efficient treatments for severe refractory patients affected by aggressive forms of the disease. Therefore, the discovery of new antigens in this context could open new perspectives for those patients. ResultsIn this study, the first objective was to improve a MS-based untargeted proteomics workflow in order to handle limited patient samples. For this purpose, a highly sensitive and robust miniaturized separation system (LC-Chip) coupled with drift tube ion mobility spectrometry and high-resolution MS was integrated in our workflow to maximize protein identification. As sample preparation can strongly influence the detectability of membrane-associated proteins, the critical steps in sample preparation were carefully optimized. As a result, 4.5 times more membrane-associated proteins were identified and experimental throughput was also drastically improved. In addition to workflow performance, particular attention was paid to assess the quality of the generated data. Indeed, several quality controls (QC) were implemented to assess data quality. Finally, the optimized workflow as well as selected QCs were evaluated in the analysis of samples containing limited number of cells. SignificanceThis work allowed the improvement of an untargeted proteomics workflow for surfaceome profiling in terms of performance. Besides, the reliability of the obtained data was evaluated through the introduction of QCs in the workflow. The applicability of the improved workflow as well as the implemented QCs for the analysis of MM primary cells obtained from patients was confirmed.

Perspective: Hidden biases in isotope delta results and the need for comprehensive reporting.

Measurements of stable-isotope composition on an isotope-delta scale can be subject to bias between laboratories or over time within a single laboratory. This bias can arise not just from differences in method protocol but also from changes in reporting guidelines, or even to the isotope-delta scales themselves. Without a clear description of method protocols, including all sample preparation steps, instrumental parameters and settings, data processing including calibration of results and estimation of measurement uncertainty, the traceability and comparability of isotope-delta values cannot be assured as bias(es) may remain hidden. To address this need, there are now clear guidelines published by IUPAC for reporting isotope-delta values for the "light" elements hydrogen, carbon, nitrogen, oxygen and sulfur.1 We recommend that authors and reviewers adhere to those guidelines when preparing and reviewing future publications.

Differentiation of Four Polyvinylidene Fluoride Polymers Based on Their End Groups by DART-FT-ICR MS and Kendrick Plots.

Nowadays, synthetic polymers are produced and used in many materials for different applications. Matrix-assisted laser desorption/ionization or electrospray mass spectrometry are classically used to investigate them, but these techniques require sample preparation steps, which are not always suitable for the study of insoluble or formulated polymers. Alternatively, direct real-time (DART) ionization analysis may be conducted without sample preparation. Four polyvinylidene fluoride (PVDF) polymers involving the C2H2F2 repeating unit coming from different suppliers have been analyzed by DART Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) in negative-ion mode. The obtained mass spectra systematically displayed an oligomeric distribution between m/z 400 and 1300 of [M - H]-, [M + O2]•-, and [M + NO2]- ions. Kendrick plots were used to ease the identification of PVDF end-groups and establish a difference between the samples. Both commercial PVDF polymers shared the same α+ω end groups formula, which confirmed a similar polymerization process for their synthesis. The two other PVDFs were clearly different from the commercial ones by the occurrence of specific end-groups. MS/MS and MS3 experiments were conducted to obtain structural information on these end-groups.

Observation of Protein Unfolding during pH Ramp Evoked by Lipase-Catalyzed Ester Hydrolysis.

Studies of protein folding often involve offline experimental methods such as titrating protein samples with denaturants or equilibrating them in the presence of denaturants. Here, we demonstrate an online analytical approach in which the protein structure is perturbed by a pH ramp evoked by immobilized lipase-catalyzed ester hydrolysis. Changes in the tertiary structure of the protein in response to a pH ramp (from approximately 6.3 to 2.8) are monitored using electrospray ionization mass spectrometry and spectrofluorometry. Interestingly, we discovered a side reaction of ammonium and formate leading to the production of cyanide that occurred during the ionization process. We also found that only certain protein analytes were bound to the formed cyanide species. Nevertheless, this problem was readily overcome by carefully selecting a specific ester substrate. Overall, the alterations in the charge-state distribution and fluorescence intensity─caused by the lipase-induced pH ramp─reveal conformational transitions in different proteins. In line with previous reports, the acid-induced denaturation of holo-myoglobin occurs through a two-step mechanism, which is supported by identification of protein-unfolding intermediates and the loss of noncovalent protein ligand (heme). The results─obtained using the developed catalytic method─are also consistent with the results of equilibrium-based experiments, while sample preparation steps are substantially reduced. The proposed approach simplifies the identification of the pH range that has the greatest impact on the protein structure. Thus, it has the potential to be a useful tool for studying protein conformational transitions in the course of pH changes.

Robust principal component analysis-multivariate adaptive regression splines (rPCA-MARS) model for determining total acid number (TAN) and total base number (TBN) of crude oil samples using attenuated total reflectance fourier transform infrared (ATR-FTIR) spectroscopy

Rapid assessments of Total Acid Number (TAN) and Total Base Number (TBN) in crude oil samples are significant in the oil industry. The analysis of TAN and TBN was conducted using the standard methods of the American Society for Testing and Materials (ASTM). However, the standard methods are costly and require a large number of samples for analysis. Therefore, providing analytical methods for the rapid crude oil analysis is very essential. In the current research, we report an application of Attenuated Total Reflection Fourier-Transform Infrared (ATR-FTIR) spectroscopy, based on robust Principal Component Analysis-Multivariate Adaptive Regression Splines (rPCA-MARS), for crude oil analysis. In the rPCA-MARS model, the ATR-FTIR spectral matrix data was decomposed into PCs scores using the rPCA method as an input data for the MARS model. The multivariate calibration models were applied to the analysis of crude oil samples based on the quantitative determination of total acid number (TAN) and total base number (TBN). An analytical method was developed for determination of TAN and TBN of crude oil samples. The ATR-FTIR spectroscopy coupled with multivariate calibration methods can be applied as a novel analytical method for crude oil samples. The result of partial least square regression (PLS-R), principle component analysis (PCR), Piecewise-Linear rPCA-MARS and Piecewise-cubic rPCA-MARS models were compared for analysis of total acid and base number of crude oil samples. The squared correlation coefficient (R2) and root mean square error (RMSE) for calibration and prediction sets were calculated to evaluate multivariate calibration models. The importance of methods was studied and discussed. The mean square error (MSE) values of Piecewise-Linear rPCA-MARS for TAN and TBN were 1.587 * 10−4 and 0.009, respectively. The Piecewise-Linear rPCA-MARS model can be successfully applied for the analysis of TAN and TBN of crude oil samples based on the obtained results. The fast and easy prediction capability of the proposed Piecewise-Linear rPCA-MARS model, as compared to standard methods, for determining TAN and TBN without any sample preparation step is important in the oil industry

A tool to assess analytical sample preparation procedures: Sample preparation metric of sustainability

Sample preparation is a key step in most analytical methods, generally regarded as the least green step of the entire procedure. The existing green metrics assess the greenness of sample preparation techniques through the evaluation of the whole analytical procedure: including sampling, sample preparation, and the final detection/quantitation. Such inclusion of the entire method makes assessing the sustainability of a newly developed sample preparation technique quite challenging, as many aspects not solely linked to the sample preparation step are unavoidably considered. Thus, an alternative metric that can explicitly and exclusively evaluate the sample preparation is proposed. The metric is simple; it reports the result with a clock-like diagram, displaying the greenness outcome of main sample preparation parameters and a total score. This new metric can differentiate closely related microextraction approaches in terms of sustainability. The metric is also open-source and can be used by downloading the Excel sheet provided.

The role of sample preparation in suspect and non-target screening for exposome analysis using human urine

The use of suspect and non-target screening (SNTS) for the characterization of the chemical exposome employing human biofluids is gaining attention. Among the biofluids, urine is one of the preferred matrices since organic xenobiotics are excreted through it after metabolization. However, achieving a consensus between selectivity (i.e. preserving as many compounds as possible) and sensitivity (i.e. minimizing matrix effects by removing interferences) at the sample preparation step is challenging. Within this context, several sample preparation approaches, including solid-phase extraction (SPE), liquid-liquid extraction (LLE), salt-assisted LLE (SALLE) and dilute-and-shoot (DS) were tested to screen not only exogenous compounds in human urine but also their phase II metabolites using liquid-chromatography coupled to high-resolution tandem mass spectrometry (LC-HRMS/MS). Additionally, enzymatic hydrolysis of phase II metabolites was evaluated. Under optimal conditions, SPE resulted in the best sample preparation approach in terms of the number of detected xenobiotics and metabolites since 97.1% of the total annotated suspects were present in samples extracted by SPE. In LLE and SALLE, pure ethyl acetate turned out to be the best extractant but fewer suspects than with SPE (80.7%) were screened. Lastly, only 52.5% of the suspects were annotated in the DS approach, showing that it could only be used to detect compounds at high concentration levels. Using pure standards, the presence of diverse xenobiotics such as parabens, industrial chemicals (benzophenone-3, caprolactam and mono-2-ethyl-5-hydroxyhexyl phthalate) and chemicals related to daily habits (caffeine, cotinine or triclosan) was confirmed. Regarding enzymatic hydrolysis, only 10 parent compounds of the 44 glucuronides were successfully annotated in the hydrolysed samples. Therefore, the screening of metabolites in non-hydrolysed samples through SNTS is the most suitable approach for exposome characterization.

A review of bioanalytical applications of microextraction techniques combined with derivatization.

Microextraction techniques have attracted the attention of many researchers working in the field of bioanalysis due to their unique advantages, mainly in downsizing the scale of sample preparation steps. In parallel, analytical derivatization offers a powerful combination in terms of additional sensitivity, selectivityand compatibility with modern separation techniques. The aim of this review is to discuss the most recent advances in bioanalytical sample preparation based on the combination of microextraction and analytical derivatization. Both innovative fundamental reports and analyte-targeted applications are included and discussed. Dispersive liquid-liquid extraction and solid-phase microextraction are the most common techniques that typically combined with derivatization, while the development of novel and greener protocols is receivingsubstantial consideration in the field of analytical chemistry.

Application of solidified floating organic droplet dispersive liquid–liquid microextraction for determination of veterinary antibiotic residues in milk samples with greenness assessment

Antibiotics are the most commonly used veterinary drug category worldwide. The extensive utilization of antibiotics in animal farming is one of the main causes of antibiotic resistance, thus posing a hazard to human health. In the current study, solidification of floating organic droplet dispersive liquid–liquid microextraction (SFO-DLLME) was developed as a green sample preparation step for extracting three widely used veterinary antibiotics; enrofloxacin, ciprofloxacin and oxytetracycline in milk samples present in the Egyptian market. SFO-DLLME was based on using 1-undecanol as a low-toxicity extracting solvent. As several effective parameters are required to be optimized in SFO-DLLME, Box–Behnken experimental design was utilized to obtain the best possible extraction results. The analysis of the analytes was performed through the development of two eco-friendly chromatographic techniques. The first method was based on HPLC-UV where the separation was achieved using C18 column with isocratic elution of mobile phase composed of 0.025 M phosphate buffer (pH 3.2): acetonitrile (85: 15 v/v) and UV detection at 278 nm. The second was UPLC-MS/MS method utilizing BEH shield C18 column acetonitrile: 0.1% formic acid in a ratio of 80:20 (v/v) as mobile phase. The two developed methods were validated it was found that they had high recoveries (88.50 %- 94%), enrichment factors(106.2 %- 112.93 %), low limit of detection (0.077 µg/kg − 29 µg/kg), and satisfactory precision RSD ≤ 2. Different brands of market milk samples were analyzed showing no residues of the studied antibiotics, while enrofloxacin was detected in a concentration of 3.74 µg/kg in raw cow milk samples. Finally, the greenness profile of the developed chromatographic methods was evaluated by the Green Analytical Procedure Index, Analytical GREEnness Metric Approach, and White Analytical Chemistry principles confirming their safety on the environment.

Monolayer magnetic nanospheres have selective binding that allow the concentration of low-abundance proteins from blood serum

Here, the efficiency of magnetic nanosphere (Fe3O4) coated with L-histidine (L-his) was evaluated to enrich proteins of low-abundance in human serum. The chemometric analysis provided optimal conditions to concentrate low-abundance proteins, reducing the dynamic range of protein levels in the serum sample. Three independent factors (temperature, mass ratio and pH) were tested. The optimal condition achieved was using temperature (42 °C), ratio (3:1, nanomaterial/sample) and pH (5.5). The kinetic study revealed that the adsorption of proteins on the surface of the monolayer magnetic nanosphere follows a pseudo-second order model with R2 = 0.9153. Adsorption isotherm experiments revealed high capacity of protein adsorption on the surface of the nanosphere, indicating a maximum theoretical adsorption capacity (qmax) of 123.45 mg g−1. The efficiency of enrichment of low-abundance proteins in the serum sample was evaluated by gel electrophoresis (SDS-PAGE). Furthermore, when compared to the method using organic solvent (acetonitrile), Fe3O4@L-his showed adequate efficiency to simplify the serum sample. Using MALDI-TOF/TOF, approximately 50 proteins were identified using Fe3O4@L-his. As a proof of concept for this study, patients with bipolar disorder and healthy controls were discriminated using the magnetic nanosphere as an adsorbent in the sample preparation step. These results show that Fe3O4@L-his can be a fast, robust, and simple alternative to enrich low-abundance proteins in human serum, emerging as an important strategy in the search for biomarkers of human diseases.

Cultivation-free sample preparation and DNA purification for direct real-time qPCR of intracellular or spore-like Coxiella burnetii in beef, goat, and lamb meat

Coxiella burnetii is a zoonotic pathogen that has been associated with foodborne outbreaks in products with ruminant origins. However, a method to detect C. burnetii in meat has been merely studied, and commercial kits cannot efficiently fulfill this purpose. In this study, an in-house preparation method for direct real-time qPCR of C. burnetii in beef, goat, and lamb meat was designed. In the sample preparation step (step 1), trypsin digestion and cell disruption techniques were introduced to target C. burnetii in an obligate intracellular or spore-like form. Afterward, 16 DNA purification protocols involving the following steps (steps 2–3) were assessed: the precipitation of meat proteins (step 2; using 2.5, 5.0 M NaCl or 1:1, 2:1 ethanol as the precipitant) and binding of DNA to silicon dioxide particles with chaotropic salts (step 3; using 2.5, 5.0 M NaCl or 2.5, 5.0 M guanidine thiocyanate as the salt). The protocols with superior performance in high-spiked loins (estimated 4–5 log cells/g) were verified in low-spiked (1–2 log cells/g) or Bacillus thuringiensis spore-inoculated (1–2 log CFU/g) loins, ribs, and hind legs. During the protein precipitation, 5.0 M NaCl induced significantly lower protein level as demonstrated by A280, when compared to 2.5 M NaCl or ethanol (P < 0.05). For the DNA binding step, Ct values were lowered in high-spiked goat or lamb loins (3.5–6.0▾; P < 0.05) when the concentration of NaCl was doubled or guanidine thiocyanate was introduced instead of NaCl as a chaotropic salt. Based on these results, two protocols using 5.0 M NaCl as the protein precipitant and 5.0 M NaCl (N2 + N2) or guanidine thiocyanate (N2 + G2) as the chaotropic salt were selected, which demonstrated successful detection in low-spiked (Ct values of N2 + N2, 32.9–35.6; N2 + G2, 32.3–36.4) or spore-inoculated meat (N2 + N2, 30.9–37.5; N2 + G2, 29.7–32.7). Verification in low-spiked meat showed that meat type/part significantly impacted the Ct values of N2 + G2 but not those of N2 + N2. To our knowledge, this is the first study that developed a highly accessible method for detecting C. burnetii in meat which could reveal the possibility of meat-borne Q fever in humans.

An eco-friendly sample preparation procedure based on air-assisted liquid-liquid microextraction for the rapid determination of phthalate metabolites in urine samples by liquid chromatography-tandem mass spectrometry

Urinary phthalate metabolite (mPAEs) analysis is a reliable tool for assessing human exposure to phthalates. With growing interest in urinary biomonitoring of these metabolites, there is a need for fast and sensitive analytical methods. Therefore, a simple, rapid procedure for simultaneous determination of fifteen phthalate metabolites in human urine samples by liquid chromatography-tandem mass spectrometry was developed. The novelty of the present procedure is based on the use of diethyl carbonate as a green biobased extraction solvent and air-assisted liquid-liquid microextraction (AALLME) as a sample preparation step. A Plackett-Burman design was used for screening the factors that influence the AALLME extraction efficiency of mPAEs. The effective factors were then optimized by response surface methodology using a central composite rotatable design. Under the optimized conditions, good linearity can be achieved in a concentration range of 1.0–20.0 ng mL-1 with correlation coefficients higher than 0.99. The repeatability and reproducibility precision were in the range of 2–12% and 1–10% respectively. Recoveries ranging from 90% to 110%. This, and the low limits of detection, ranging from 0.01 to 0.05 ng mL-1, make the proposed procedure sensitive and suitable for human biomonitoring of phthalate exposures. For proof-of-principle, the new method was used to measure the urinary concentrations of mPAEs in 20 urine samples from Brazilian women. The high frequency of detections and in part high concentrations of mPAEs indicate to widespread exposure to several phthalates among Brazilian women.

Is this the end of dried blood spots as we know it?

In 2017 integrated sampling and sample preparation for simplified liquid chromatography-mass spectrometry analysis of proteins from dried blood spots were introduced. The concept, called smart samplers or smart sampling, enables proteolysis or affinity clean-up, two common sample preparation steps in liquid chromatography-mass spectrometric bioanalysis of proteins, to start at the moment of sampling. The idea is to utilize the time for sampling and drying to perform these time-consuming and labour-intensive steps. Hence, only a simplified sample preparation is necessary after the arrival of the sample in the lab. In this perspective, we present an overview of the smart sampling approach where the conventional protein analysis workflow is reshuffled to start already prior to arrival in the lab. In addition, we present a thorough discussion of integrating sample preparation steps such as proteolysis or affinity capture in the sampling. Finally, in the end, we try to answer the question if conventional dried blood spots will become obsolete in the future.

Evaluating cPILOT Data toward Quality Control Implementation.

Multiplexing enables the monitoring of hundreds to thousands of proteins in quantitative proteomics analyses and increases sample throughput. In most mass-spectrometry-based proteomics workflows, multiplexing is achieved by labeling biological samples with heavy isotopes via precursor isotopic labeling or isobaric tagging. Enhanced multiplexing strategies, such as combined precursor isotopic labeling and isobaric tagging (cPILOT), combine multiple technologies to afford an even higher sample throughput. Critical to enhanced multiplexing analyses is ensuring that analytical performance is optimal and that missingness of sample channels is minimized. Automation of sample preparation steps and use of quality control (QC) metrics can be incorporated into multiplexing analyses and reduce the likelihood of missing information, thus maximizing the amount of usable quantitative data. Here, we implemented QC metrics previously developed in our laboratory to evaluate a 36-plex cPILOT experiment that encompassed 144 mouse samples of various tissue types, time points, genotypes, and biological replicates. The evaluation focuses on the use of a sample pool generated from all samples in the experiment to monitor the daily instrument performance and to provide a means for data normalization across sample batches. Our results show that tracking QC metrics enabled the quantification of ∼7000 proteins in each sample batch, of which ∼70% had minimal missing values across up to 36 sample channels. Implementation of QC metrics for future cPILOT studies as well as other enhanced multiplexing strategies will help yield high-quality data sets.

Comparison of Quantitative Mass Spectrometric Methods for Drug Target Identification by Thermal Proteome Profiling.

Thermal proteome profiling (TPP) provides a powerful approach to studying proteome-wide interactions of small therapeutic molecules and their target and off-target proteins, complementing phenotypic-based drug screens. Detecting differences in thermal stability due to target engagement requires high quantitative accuracy and consistent detection. Isobaric tandem mass tags (TMTs) are used to multiplex samples and increase quantification precision in TPP analysis by data-dependent acquisition (DDA). However, advances in data-independent acquisition (DIA) can provide higher sensitivity and protein coverage with reduced costs and sample preparation steps. Herein, we explored the performance of different DIA-based label-free quantification approaches compared to TMT-DDA for thermal shift quantitation. Acute myeloid leukemia cells were treated with losmapimod, a known inhibitor of MAPK14 (p38α). Label-free DIA approaches, and particularly the library-free mode in DIA-NN, were comparable of TMT-DDA in their ability to detect target engagement of losmapimod with MAPK14 and one of its downstream targets, MAPKAPK3. Using DIA for thermal shift quantitation is a cost-effective alternative to labeled quantitation in the TPP pipeline.

Conductivity-based approach to estimate average bundle length in randomly oriented network of single-walled carbon nanotubes

In this paper, we report a method to estimate the average length of bundles in a randomly oriented network of single-walled carbon nanotubes (SWCNTs) by analyzing the dependence of its sheet resistance on the distance between contacts. We propose an analytical model to verify the method and find the model's limitations using Monte Carlo simulations. Then, we apply the proposed approach to experimental results acquired from 95% (at 550 nm wavelength) transparent SWCNT films. The proposed method to estimate the average SWCNT bundle length can be used in situ and does not require any specific sample preparation step that can distort the SWCNT network.

Dilute-and-shoot coupled to mixed mode liquid chromatography-tandem mass spectrometry for the analysis of persistent and mobile organic compounds in human urine

In this work, a comprehensive method for the simultaneous determination of 33 diverse persistent and mobile organic compounds (PMOCs) in human urine was developed by dilute-and-shoot (DS) followed by mixed-mode liquid chromatography coupled with tandem mass spectrometry (MMLC-MS/MS). In the sample preparation step, DS was chosen since it allowed the quantification of all targets in comparison to lyophilization. For the chromatographic separation, Acclaim Trinity P1 and P2 trimodal columns provided greater capacity for retaining PMOCs than reverse phase and hydrophilic interaction liquid chromatography. Therefore, DS was validated at 5 and 50 ng/mL in urine with both mixed mode columns at pH = 3 and 7. Regarding figures of merit, linear calibration curves (r2 > 0.999) built between instrumental quantification limits (mostly below 5 ng/mL) and 500 ng/mL were achieved. Despite only 60% of the targets were recovered at 5 ng/mL because of the dilution, all PMOCs were quantified at 50 ng/mL. Using surrogate correction, apparent recoveries in the 70–130% range were obtained for 91% of the targets. To analyse human urine samples, the Acclaim Trinity P1 column at pH = 3 and 7 was selected as a consensus between analytical coverage (i.e. 94% of the targets) and chromatographic runs. In a pooled urine sample, industrial chemicals (acrylamide and bisphenol S), biocides and their metabolites (2-methyl-4-isothiazolin-3-one, dimethyl phosphate, 6-chloropyridine-3-carboxylic acid, and ammonium glufosinate) and an artificial sweetener (aspartame) were determined at ng/mL levels. The outcomes of this work showed that humans are also exposed to PMOCs due to their persistence and mobility, and therefore, further human risk assessment is needed.

Portable biosensor for the detection of Enrofloxacin and Ciprofloxacin antibiotic residues in food, body fluids, environmental and wastewater samples

Enrofloxacin (ENR) and its metabolite Ciprofloxacin (CIP) are both a class of fluoroquinolone antibiotics effective against a broad-spectrum microbial infection. Recent surge in the consumption of CIP and ENR has been linked to increased cases of drug-resistant pathogens. This is due to the fact that the antibiotic residues remain in milk, meat, soil and environmental water for a prolonged duration. Although gold standard methods such as LC-MS are sensitive, they suffer from expensive operation and maintenance cost, and would need dedicated facilities and tedious sample preparation steps. Such limitations make on site detection impossible for regulatory bodies in developing countries. To address this issue, we developed a portable device that can detect the presence of CIP and ENR antibiotics in the range of parts per billion (ppb) concentrations accurately. It consists of a polyaniline (PAni) coated U-bent optical fiber with anti-ENR/CIP antibody immobilized on the polymer surface. The sensor relies on the principle of evanescent wave absorbance by antigen-antibody complex. The sensor showed limit of detection (LOD) of 1 ppb with a linear range of operation from 1 ppb to 500 ppb (R2 = 0.96-0.99) in lake water, waste water treatment plant effluent, urine, blood serum, milk and meat samples. The recovery of the sensor ranges from 88% to 120% indicating reasonable accuracy. The sensor has excellent selectivity towards CIP and ENR and showed stability for four weeks indicating its field deployability and robustness. The portable sensor is scalable and contract has been given to an industry partner to mass manufacture the device.

Capillary surface modification using millimolar levels of aminosilane reagent for highly efficient separation of phenolic acids and flavonols by capillary electrophoresis with UV detection.

Phytochemical analysis of phenolic acids and flavonols poses a challenge, necessitating the development of an efficient separation method. This facilitates the quantification of these compounds, yielding valuable insights into their benefits. To develop a highly effective separation of phenolic acids and flavonols by capillary electrophoresis and ultraviolet (UV) detection through the modification of the capillary surface using 3-aminopropyltriethoxysilane (APTES) at millimolar concentrations. The capillary surface is modified with 0.36 mM-APTES solution. The electrolyte is 20.0 mM borate buffer (pH9.0). Separation performance (plate number N, resolution Rs ), stability, and reproducibility of the coating procedure are evaluated using the analysis of phenolic acids, rutin and quercetin. The modified capillary provided efficient separation with plate numbers N ≥ 1.0 × 104 m-1 and resolution Rs ≥ 0.8 for all pairs of adjacent peaks of the separation of five selected phenolic acids, rutin, quercetin, caffeine and methylparaben (as internal standard). The precisions of the relative migration times for 17 consecutive analyses of samples over 3h were 1% relative standard deviation (RSD) for rutin and 7% RSD for quercetin. The analysis of rutin and quercetin in 12 dietary supplement product samples only required a simple dilution step for sample preparation. A straightforward modification technique utilising millimolar concentrations of APTES resulted in highly efficient separation of phenolic acids, rutin and quercetin, accompanied by high precision and surface stability. The modified capillary proved successful in analysing rutin and quercetin content in dietary supplements.

Extraction of ibuprofen from baby lettuces by natural deep eutectic systems

Vegetable crops can adsorb hazardous pollutants from water and soils. The occurrence of pharmaceuticals in the environment is considered a problematic matter with unknown long-term effects. Thus, the objective of this work was to develop, for the first time, a rapid, sensitive, and ecological methodology based on Natural Deep Eutectic Systems (NADES) to extract ibuprofen in baby lettuce samples. The proposed strategy involves a sustainable sample preparation step, leading to an efficient and selective analytical methodology suitable for high-throughput analysis (<3 samples min). NADES were designed and physicochemically characterized. The effect of experimental variables on extraction selectivity and efficiency: NADES composition, sample/solvent ratio, and extraction time and temperature were studied and optimized, resulting in 0.1 g of dry sample, 1 mL of choline chloride:glycerol 1:5, and 10 min of extraction at 80 °C. The limits of detection and quantification were 2.74 and 8.31 ng g−1 (dry weight), respectively; with a linear range of 8.31–4000 ng g−1 dw. The recovery % and RSD % were 98.1–99.6% and < 2.97%, respectively. The methodology and the sample preparation show excellent green performance using the greenness calculator, obtaining scores of 0.68 and 0.83. Finally, the ibuprofen uptake study revealed that baby lettuces adsorb ibuprofen at high concentration levels.