Analysis of highly polar anionic pesticides in food of plant and animal origin by ion chromatography and tandem mass spectrometry with emphasis on addressing adverse effects caused by matrix co-extractives.

Residues of various highly polar pesticides and their metabolites are commonly found in numerous food products. Some of these compounds, such as glyphosate, are not only used in large amounts in agriculture, but are also controversially discussed in public. Here, we present a method, employing ion chromatography (IC) coupled to tandem mass spectrometry (IC-MS/MS), for the analyses of glyphosate, aminomethyl phosphonic acid (AMPA), N-acetyl-glyphosate (NAGly), fosetyl, and 10 further highly polar pesticides and metabolites in various plant and animal matrices following a minimal sample preparation by means of the QuPPe method. Thorough investigations showed that an AS19 column enabled the analysis of all 14 compounds within 30 min. The best sensitivity could be obtained with the make-up solvent acetonitrile being admixed to the mobile phase at a 1:2 flow rate ratio. Matrix effects were thoroughly studied in terms of ion suppression and retention time shifts. Conductivity detection was used to monitor elution profiles of matrix co-extractives in comparison with matrix effect profiles obtained by continuous post-column infusion of a mix with 13 highly polar pesticides and metabolites. These tests indicated that a fivefold dilution of QuPPe extracts was suitable for the routine analysis of samples for MRL-conformity, as it considerably reduced matrix effects maintaining sufficient sensitivity and high recovery rates in eight different commodities. The suitability of the final method for its application in routine analysis was verified by the analysis of >130 samples containing incurred residues where the results were compared with two existing LC-MS/MS methods.

Utilization of Matrix Effect for Enhancing Resolution in Cation Exchange Chromatography.

In ion chromatography studies, the matrix effect of other inorganic ions present in the sample is a well-known phenomenon. In this work, the behavior of inorganic and organic ions was studied in a system overloaded with ammonium ions. The ammonium ions came from a solution of ammonium hydroxide in various concentrations (0.25-1.25%). In this system, which was significantly overloaded with ammonium ions, the behavior of three ions were tested (lithium, tris, and sodium cations). The measurements were performed at different eluent concentrations (6-17 mM), chromatographic column temperatures (25-40 °C), and injected volumes (15-40 µL). The retention times of sodium and lithium ions increased with increasing amounts of injected ammonium, while tris remained essentially unchanged, indicating that the resolution of these ions can be influenced by varying the concentration of the matrix. The results suggested that the observed effect was due to a combination of the pH change caused by the injected matrix, the dissociation of tris ions, the dissociation of the carbocylic ion-exchange groups of stationary phase, the change in buffer capacity, and the amount of ammonium ion introduced. It has been shown that in a well-designed experiment, the addition of ammonium hydroxide to the sample at concentrations greater than 1% can improve the efficiency of organic and inorganic cation separation. It was found that 8 mM methanesulfonic acid eluent, 30 °C, 1% ammonium hydroxide matrix concentration, and 25 µL injection were optimal for the baseline separation of tris and sodium ions on the high-capacity Dionex CS16 column. These ions could not be separated on this column without the presence of the ammonium matrix.

Evaluation of an Oral Fluid Collection Device and a Solid-Phase Extraction Method for the Determination of Coca Leaf Alkaloids by Gas Chromatography-Mass Spectrometry.

Some South American countries have ancient traditions that may pose legal problems, such as the consumption of coca leaves, as this can provide positive results for cocaine use after the analysis of biological samples. For this reason, it is necessary to find specific markers that help differentiate legal from illegal consumption, such as tropacocaine, cinnamoylcocaine, and especially hygrine and cuscohygrine. In this work, two techniques for collecting biological samples are compared: the Quantisal® Oral Fluid collection device and passive drooling. Once the samples were collected, they were subjected to solid-phase extraction for subsequent injection into GC-MS. Different validation parameters included in international guides have been studied to evaluate whether the proposed method is valid for the defined purpose, placing special emphasis on the study of the matrix effect and little value on GC-MS analyses. With respect to this parameter, an increase in the signal was found for CUS and t-CIN, but it was not significant for the rest of the substances studied. The recoveries have varied significantly depending on the way of working, being higher when working with standardized areas. After carrying out work with the oral fluid samples collected from laboratory volunteers, the method was applied to two real samples. The results obtained support the need for further research to overcome certain limitations presented by the device.

From tyrosine to hydroxytyrosol: a pathway involving biologically active compounds and their determination in wines by ultra performance liquid chromatography with mass spectrometry.

BACKGROUND: Hydroxytyrosol (HT) is a bioactive compound present in a limited number of foods such as wines, olives, and olive oils. During alcoholic fermentation, yeast converts aromatic amino acids into higher alcohols such as tyrosol, which can undergo hydroxylation into HT. The aim of this study was to validate an analytical method using ultra performance liquid chromatography coupled with mass spectrometry (UPLC/MS-MS) to quantify HT and its precursors (tyrosine, hydroxyphenylpyruvic acid, hydroxyphenylacetaldehyde, 4-hydroxyphenylacetic acid, and tyrosol) in wines. Their occurrence was evaluated in a total of 108 commercial Spanish wine samples. RESULTS: The validated method simultaneously determined both HT and its precursors, with adequate limits of detection between 0.065 and 21.86 ng mL-1 and quantification limits between 0.199 and 66.27 ng mL-1 in a 5 min run. The concentration of HT in red wines was significantly higher (0.12-2.24 mg L-1) than in white wines (0.01-1.27 mg L-1). The higher the alcoholic degree, the higher was the content of HT. The bioactive 4-hydroxyphenylacetic acid was identified in Spanish wines for the first time at 3.90-127.47 mg L-1, being present in all the samples. CONCLUSION: The highest HT concentrations were found in red wines and in wines with higher ethanol content. These data are useful for a further estimation of the intake of these bioactive compounds and to enlarge knowledge on chemical composition of wines. © 2024 The Author(s). Journal of the Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Optimizing the QuEChERS method for efficient monitoring of fipronil, thiobencarb, and cartap residues in paddy soils with varying properties.

This study aims to optimize the QuEChERS methodology for extracting three pesticides (fipronil, thiobencarb, and cartap) from two paddy soils with distinct characteristics. Various modifications were explored to enhance extraction efficiency, employing acetonitrile (MeCN) or ethyl acetate (EtOAc) for extraction and primary-secondary amine (PSA) and graphitized carbon black (GCB) for the clean-up. Assessment criteria included accuracy, precision, linearity, detection limits, uncertainty, and matrix effects. Results revealed that the clayey soil with lower organic carbon (OC) content (1.26%) and 100% moisture yielded the highest pesticide recoveries (113.72%, 115.73%, and 116.41% for FIP, THIO, and CART, respectively). In contrast, the silty clayey soil with higher OC content (2.91%) and 20% water content exhibited poor recoveries (< 60%). FIP and CART demonstrated better recoveries with MeCN, while THIO performed better with EtOAc under specific moisture conditions. Clean-up sorbents significantly reduced FIP and CART recoveries, with THIO recoveries less affected. Acidifying with HCl substantially improved CART recovery. EtOAc introduced a moderate to strong matrix effect for FIP and THIO, while MeCN in soils with 100% moisture resulted in a strong matrix effect for CART. The study highlighted the substantial impact of extraction conditions, pesticide properties, and soil conditions on the outcomes of the QuEChERS method. A comprehensive understanding of these interplays was deemed crucial for accurately quantifying pesticide residues in agricultural soils.

Organochlorine pesticides in vegetable oils: An overview of occurrence, toxicity, and chromatographic determination in the past twenty-two years (2000-2022).

Organochlorine pesticides (OCPs) are used globally to control pests in the food industry. However, some have been banned due to their toxicity. Although they have been banned, OCPs are still discharged into the environment and persist for long periods of time. Therefore, this review focused on the occurrence, toxicity, and chromatographic determination of OCPs in vegetable oils over the last 22 years (2000-2022) (111 references).Literature search shows that OCPs kill pests by destroying endocrine, teratogenic, neuroendocrine, immune, and reproductive systems. However, only five studies investigated the fate of OCPs in vegetable oils and the outcome revealed that some of the steps involved during oil processing introduce more OCPs. Moreover, direct chromatographic determination of OCPs was mostly performed using online LC-GC methods fitted with oven transfer adsorption desorption interface. While indirect chromatographic determination was favored by QuEChERS extraction technique, gas chromatography frequently coupled to electron capture detection (ECD), gas chromatography in selective ion monitoring mode (SIM), and gas chromatography tandem mass spectrometry (GC-MS/MS) were the most common techniques used for detection. However, the greatest challenge still faced by analytical chemists is to obtain clean extracts with acceptable extraction recoveries (70-120%). Hence, more research is still required to develop greener and selective extraction methods toward OCPs, thus improving extraction recoveries. Moreover, advanced techniques like gas chromatography high resolution mass spectrometry (GC-HRMS) must also be explored. OCPs prevalence in vegetable oils varied greatly in various countries, and concentrations of up to 1500 µg/kg were reported. Additionally, the percentage of positive samples ranged from 1.1 to 97.5% for endosulfan sulfate.

Deciphering the human urine matrix: a new approach to simultaneously quantify the main ions and organic compounds by ion chromatography/mass spectrometry (IC-MS).

Analyzing the composition of (human) urine plays a major role in the fields of biology and medicine. Organic molecules (such as urea, creatine) and ions (such as chloride, sulfate) are the major compounds present in urine, the quantification of which allows for the diagnosis of a subject's health condition. Various analytical methods have been reported for studying urine components and validated on the basis of known and referenced compounds. The present work introduces a new method able to simultaneously determine both major organic molecules and ions contained in urine, by combining ion chromatography using a conductimetric detector with mass spectroscopy. The analysis of organic and ionized compounds (anionic and cationic) was achieved in double injections. For quantification, the standard addition method was used. Human urine samples were pre-treated (diluted and filtered) for IC-CD/MS analysis. The analytes were separated in 35 min. Calibration ranges (0-20 mg.L-1) and correlation coefficients (> 99.3%) as well as detection (LODs < 0.75 mg.L-1) and quantification (LOQs < 2.59 mg.L-1) limits were obtained for the main organic molecules (lactic, hippuric, citric, uric, oxalic acids, urea, creatine, and creatinine) and ions (chloride, sulfate, phosphate, sodium, ammonium, potassium, calcium, and magnesium) contained in urine. The intra- and inter-day accuracies of the analytes consistently ranged from 0.1 to 5.0%, and the precision was within 4.0%. For all analytes, no significant matrix effects were observed, and recoveries ranged from 94.9 to 102.6%. Finally, quantitative results of analytes were obtained from 10 different human urine samples.

Preparation of 18O-labelled azaspiracids for accurate quantitation using liquid chromatography-mass spectrometry.

Azaspiracids (AZAs) are a group of polyether marine algal toxins known to accumulate in shellfish, posing a risk to human health and the seafood industry. Analysis of AZAs is typically performed using LC-MS, which can suffer from matrix effects that significantly impact the accuracy of measurement results. While the use of isotopic internal standards is an effective approach to correct for these effects, isotopically labelled standards for AZAs are not currently available. In this study, 18O-labelled AZA1, AZA2, and AZA3 were prepared by reaction with H218O under acidic conditions, and the reaction kinetics and sites of incorporation were studied using LC-HRMS/MS aided by mathematical analysis of their isotope patterns. Analysis of the isotopic incorporation in AZA1 and AZA3 indicated the presence of four exchangeable oxygen atoms. Excessive isomerization occurred during preparation of 18O-labelled AZA2, suggesting a role for the 8-methyl group in the thermodynamic stability of AZAs. Neutralized mixtures of 18O-labelled AZA1 and AZA3 were found to maintain their isotopic and isomeric integrities when stored at -20 °C and were used to develop an isotope-dilution LC-MS method which was applied to reference materials of shellfish matrices containing AZAs, demonstrating high accuracy and excellent reproducibility. Preparation of isotopically labelled compounds using the isotopic exchange method, combined with the kinetic analysis, offers a feasible way to obtain isotopically labelled internal standards for a wide variety of biomolecules to support reliable quantitation.

Removal of contaminants of emerging concern from the supernatant of anaerobically digested sludge by O3 and O3/H2O2: Ozone requirements, effects of the matrix, and toxicity.

Digestate is a rich source of nutrients that can be applied in agricultural fields as fertilizer or irrigation water. However, most of the research about application of digestate have focused on its agronomic properties and neglected the potential harm of the presence of contaminants of emerging concern (CECs). Aadvanced oxidation processes (AOPs) have proved to be effective for removing these compounds from drinking water, yet there are some constrains to treat wastewater and digestate mainly due to their complex matrix. In this study, the feasibility to remove different CECs from digestate using O3 and O3/H2O2 was assessed, and the general effect of the matrix in the oxidation was explained. While the lab-scale ozonation provided an ozone dose of 1.49 mg O3/mg DOC in 5 h treatment, almost all the compounds were removed at a lower ozone dose of maximum 0.48 mg O3/mg DOC; only ibuprofen required a higher dose of 1.1 mg O3/mg DOC to be oxidized. The digestate matrix slowed down the kinetic ozonation rate to approximately 1% compared to the removal rate in demineralized water. The combined treatment (O3/H2O2) showed the additional contribution of H2O2 by decreasing the ozone demand by 59-75% for all the compounds. The acute toxicity of the digestate, measured by the inhibition of Vibrio fisheries luminescence, decreased by 18.1% during 5 h ozonation, and by 34% during 5 h O3/H2O2 treatment. Despite the high ozone consumption, the ozone dose (mg O3/mg DOC) required to remove all CECs from digestate supernatant was in the range or lower than what has been reported for other (waste-)water matrix, implying that ozonation can be considered as a post-AD treatment to produce cleaner stream for agricultural purposes.

A simplified D3 -creatine dilution method for skeletal muscle mass determination with dynamic correction of creatinine and D3 -creatinine using ultra-performance liquid chromatography-tandem mass spectrometry.

This study developed a simple method for muscle mass determination based on D3 -creatine dilution by removing the matrix effects of ultra-performance liquid chromatography-tandem mass spectrometry analysis through mutual correction of creatinine and D3 -creatinine. Rats were administered an oral tracer dose of D3 -creatine at age 6 weeks. Creatinine and D3 -creatinine in urine were detected using ultra-performance liquid chromatography-tandem mass spectrometry after diluting 20 times to obtain D3 -creatinine enrichment factor (mole percent excess). The mole percent excess obtained from peak area could be used to calculate muscle mass using the improved formula. The limit of detection was 0.500 ng/mL for D3 -creatinine. Creatinine and D3 -creatinine could be mutually corrected because of the same matrix effect, and D3 -creatine spillage was negligible within 0.22%. Isotopic steady time was consistent with that obtained using conventional methods. Bland-Altman plots demonstrated the satisfying consistency between the proposed method and magnetic resonance imaging. This is a simple and rapid measuring method of muscle mass based on D3 -creatine dilution that requires no accurate quantification of creatinine and D3 -creatinine concentrations and no urine sample collection to obtain D3 -creatine spillage.

Use of an experimental design to optimise the saponification reaction and the quantification of vitamins A1 and A2 in whole fish.

In ASEAN countries, small freshwater fish species contribute to the nutritional needs of people with few livelihoods by providing them with significant amounts of protein, fat, vitamins and minerals. Some species are eaten whole (with their organs, skin, bones, head and eyes). To estimate the vitamin A content of these foods, conventional saponification has been applied but has not been able to fully release the retinol. Our objective was to optimise the conditions of vitamin A saponification in whole fish to have a reliable estimate of their contribution to intakes. The effects of temperature and saponification time on the retinol quantification of whole fish were evaluated using a two-factor experimental design. Reaction time had a significant effect on the saponification of standard retinyl palmitate and whole fish (p≤0.05). For whole fish, the best conditions for the saponification were to heat the samples to 80 °C for 43 minutes. Under these conditions, the retinol is well liberated from the matrix and protected from degradation and isomerisation reactions. The time-temperature couple used is more intense than that recommended for quantifying vitamin A in milk or enriched margarines. The protective effect of the food matrix against the release of retinol is evident. Vitamin A2 alcohol (3,4-didehydroretinol) was detected in five species and the overall vitamin A contents ranged from 9.6 to 737.5 µg RE/100 g in species frequently consumed in Cambodia. The two species of small fish consumed whole were the ones that contained significantly more vitamin A among the ten tested (p≤0.05). Highlights: Vitamin A2 alcohol was quantified in five fish species. The official saponification partially released retinol in whole fish. The optimised reaction required heating the sample to 80 °C for 43 min.

Removal of multiple lipids from human plasma using a hydroxyl-functionalized covalent organic framework aerogel as a new sorbent.

A hydroxyl-functionalized covalent organic framework aerogel COFTHB-TAPB-aerogel was designed and prepared as an adsorbent for the removal of multiple lipids from human plasma. The applications of 1,3,5-tris(4'-hydroxy-5'-formylphenyl)benzene (THB) and 1,3,5-tris(4-aminophenyl)benzene (TAPB) as monomers, DMSO/mesitylene (v/v, 4/1) as reaction solvent, and n-propylamine as reaction regulator endow COFTHB-TAPB-aerogel with good adsorption performance for multiple lipids. The morphology, phase purity, specific surface area, pore size, surface charge, and stability of COFTHB-TAPB-aerogel were characterized. Adsorption thermodynamics and adsorption kinetics studies showed that COFTHB-TAPB-aerogel had high equilibrium adsorption capacities (> 15913 mg g-1) and fast adsorption equilibrium (≤ 10 s) for the four model lipids tested. COFTHB-TAPB-aerogel had good reusability with the removal of the model lipids being still more than 91% after 10 use cycles. The sample pretreatment conditions and adsorbent amounts used in lipids removal experiments were optimized. Under the optimized conditions, the method of ultra-high performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) using COFTHB-TAPB-aerogel as solid-phase extraction sorbent was validated with negligible matrix effects (0.4-3.0%) and good accuracy (86.7-110%) and was applied to determine  20 amino acids in human plasma samples from healthy individuals and gastric adenocarcinoma (GA) patients. The established method has been proved to have good application potential for the removal of multiple lipids in human plasma to reduce the matrix effects and improve the accuracy of clinical LC-MS analysis.

A Proposed Methodology to Deal with the Impact of In Vitro Cellular Matrix on the Analytical Performances of a Targeted Metabolomic LC-HRMS Method.

Performances of metabolomic methods have been widely studied on biological matrices such as serum, plasma, and urine; but much less on in vitro cell extracts. While the impact of cell culture and sample preparation on results are well-described, the specific effect of the in vitro cellular matrix on the analytical performance remains uncertain. The aim of the present work was to study the impact of this matrix on the analytical performance of an LC-HRMS metabolomic method. For this purpose, experiments were performed on total extracts from two cell lines (MDA-MB-231 and HepaRG) using different cell numbers. Matrix effects, carryover, linearity, and variability of the method were studied. Results showed that the performances of the method depend on the nature of the endogenous metabolite, the cell number, and the nature of the cell line. These three parameters should, therefore, be considered for the processing of experiments and the interpretation of results depending on whether the study focuses on a limited number of metabolites or aims to establish a metabolic signature.

Implication of the Polymeric Phenolic Fraction and Matrix Effect on the Antioxidant Activity, Bioaccessibility, and Bioavailability of Grape Stem Extracts.

The bioaccessibility and bioavailability of phenolics compounds of two grape stem extracts with different composition were studied. High polymeric extract (HPE) presented a higher content of total phenolics (TPC), procyanidins, hemicelluloses, proteins, and ashes, whereas low procyanidin extract (LPE) showed a higher fat, soluble sugars, and individual phenolic compounds content. Corresponding to its higher total phenolics content, HPE possesses a higher antioxidant activity (TEAC value). The digestion process reduced the antioxidant activity of the HPE up to 69%, due to the decrease of TPC (75%) with a significant loss of polymeric compounds. LPE antioxidant activity was stable, and TPC decreased by only 13% during the digestion process. Moreover, a higher antioxidant phenolic compounds bioavailability was shown in LPE in contrast to HPE. This behaviour was ascribed mainly to the negative interaction of polymeric fractions and the positive interaction of lipids with phenolic compounds. Therefore, this study highlights the convenience of carrying out previous studies to identify the better extraction conditions of individual bioavailable phenolic compounds with antioxidant activity, along with those constituents that could increase their bioaccessibility and bioavailability, such as lipids, although the role played by other components, such as hemicelluloses, cannot be ruled out.

Validation and Simultaneous Monitoring of 311 Pesticide Residues in Loamy Sand Agricultural Soils by LC-MS/MS and GC-MS/MS, Combined with QuEChERS-Based Extraction.

Soil can be contaminated by pesticide residues through agricultural practices, by direct application or through spray-drift in cultivations. The dissipation of those chemicals in the soil may pose risks to the environment and human health. A simple and sensitive multi-residue analytical method was optimized and validated for the simultaneous determination of 311 active substances of pesticides in agricultural soils. The method involves sample preparation with QuEChERS-based extraction, and determination of the analytes with a combination of GC-MS/MS and LC-MS/MS techniques. Calibration plots were linear for both detectors over the range of five concentration levels, using matrix-matched calibration standards. The obtained recoveries from fortified-soil samples ranged from 70 to 119% and from 72.6 to 119% for GC-MS/MS and LC-MS/MS, respectively, while precision values were <20% in all cases. As regards the matrix effect (ME), signal suppression was observed in the liquid chromatography (LC)-amenable compounds, which was further estimated to be negligible. The gas chromatography (GC)-amenable compounds showed enhancement in the chromatographic response estimated as medium or strong ME. The calibrated limit of quantification (LOQ) value was 0.01 µg g-1 dry weight for most of the analytes, while the corresponding calculated limit of determination (LOD) value was 0.003 µg g-1 d.w. The proposed method was subsequently applied to agricultural soils from Greece, and positive determinations were obtained, among which were non-authorized compounds. The results indicate that the developed multi-residue method is fit for the purpose of analyzing low levels of pesticides in soil, according to EU requirements.

Pesticide residues in agricultural end-products and risk assessment for consumers in North China.

This study investigated pesticide residues in market-sold vegetables and fruits in Hebei Province, China, over 5 years (2018-2022). A modified QuEChERS (Quick, Easy, Cheap, Effective, Rugged, and Safe) method was applied to gas chromatography with triple-quadrupole mass spectrometer (GC-MS/MS). The analytical methods were validated with respect to matrix effect (ME), recovery rate (78.9~105.5%), limit of quantitation (LOQ, 2.93~9.73 µg/kg), and linear correlation coefficient (0.9982~0.9997). Residues of 10 pesticides in 12 categories of vegetable and fruit were detected. 31.9% of the samples were detected pesticide residues; 15.5% of samples were detected multi-component pesticide residues. Twenty-seven positive detections of pesticide residues exceeded the corresponding maximum residue limit (MRL), accounting for 2.33%. The most types of pesticide residues were detected in cherry, with the number of 7. Procymidone was the most detected pesticide, and it was detected in 8 categories of samples. The hazard index (HI) of omethoate was the highest and the procymidone was the lowest. The HI of all the vegetables and fruits were less than 100%. The effects of pesticide residues are within an acceptable range for human. Adequate attention and further monitoring are still needed.

The Matrix Effect on Nonlinear Optical Responses of Disperse Orange 25: Optical Bistability and Z-Scan.

The nonlinear behaviours of an azo dye, Disperse Orange 25 (DO25), doped on two polymers (PVP and PMMA), with different weight percentages are investigated under irradiation of 300mW continuous Nd-YAG Laser (λ = 532 nm). The optical bistability (OB) of samples was examined using the Mach-Zehnder interferometer. The obtained results show that the PVP matrix doped with DO25 has better nonlinear responses due to their efficiency and stability depend on the properties of the matrix such as the glass transition temperature which is higher for PVP and the structural properties. Also, the third-order refractive index and nonlinear absorption coefficient are measured by (CA) and (OA) Z-scan techniques, respectively.

Complement C1q in plasma induces nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticle antibody conjugates.

Upconverting nanoparticles are attractive reporters for immunoassays, because their high specific activity and lack of autofluorescence background enable their detection at extremely low concentrations. However, the sensitivity achieved with heterogeneous sandwich immunoassays using nanoparticle reporters is generally limited by the nonspecific binding of nanoparticle antibody conjugates to solid supports. In this study, we characterized plasma components associated with elevated nonspecific binding of poly(acrylic acid)-coated upconverting nanoparticles in heterogeneous two-step sandwich immunoassays. Plasma was consecutively fractionated using various chromatographic methods by selecting after each step the fractions producing the highest nonspecific binding of upconverting nanoparticle conjugates in an immunoassay for cardiac troponin I. Finally, the proteins in the fractions associated with highest amount of nonspecific binding were separated by gel electrophoresis and identified with mass spectrometry. The results indicated that complement component C1q was present in the fractions associated with the highest signal from nonspecific binding. The interference was not limited to only poly(acrylic acid)-coated nanoparticles or certain antibody combination, but occurred more generally. The interference was removed by increasing the ionic strength of the assay buffer in the sample incubation step or by adding a negatively charged blocker to bind on positively charged C1q, suggesting that the interaction is mostly electrostatic. Hence, we assume that the interference is likely to affect various negatively charged nanoparticles. The identification of complement component C1q as the major interfering protein allows for more rational design of countermeasures in future immunoassay development utilizing nanoparticle reporters.

Blood biomarkers in ALS: challenges, applications and novel frontiers.

Amyotrophic lateral sclerosis (ALS) is the most common motor neuron disease among adults. With diagnosis reached relatively late into the disease process, extensive motor cell loss narrows the window for therapeutic opportunities. Clinical heterogeneity in ALS and the lack of disease-specific biomarkers have so far led to large-sized clinical trials with long follow-up needed to define clinical outcomes. In advanced ALS patients, there is presently limited scope to use imaging or invasive cerebrospinal fluid (CSF) collection as a source of disease biomarkers. The development of more patient-friendly and accessible blood biomarker assays is hampered by analytical hurdles like the matrix effect of blood components. However, blood also provides the opportunity to identify disease-specific adaptive changes of the stoichiometry and conformation of target proteins and the endogenous immunological response to low-abundance brain peptides, such as neurofilaments (Nf). Among those biomarkers under investigation in ALS, the change in concentration before or after diagnosis of Nf has been shown to aid prognostication and to allow the a priori stratification of ALS patients into smaller sized and clinically more homogeneous cohorts, supporting more affordable clinical trials. Here, we discuss the technical hurdles affecting reproducible and sensitive biomarker measurement in blood. We also summarize the state of the art of non-CSF biomarkers in the study of prognosis, disease progression, and treatment response. We will then address the potential as disease-specific biomarkers of the newly discovered cryptic peptides which are formed down-stream of TDP-43 loss of function, the hallmark of ALS pathobiology.

Estimation of the concentrations of hydroxylated polychlorinated biphenyls in human serum using ionization efficiency prediction for electrospray.

Hydroxylated PCBs are an important class of metabolites of the widely distributed environmental contaminants polychlorinated biphenyls (PCBs). However, the absence of authentic standards is often a limitation when subject to detection, identification, and quantification. Recently, new strategies to quantify compounds detected with non-targeted LC/ESI/HRMS based on predicted ionization efficiency values have emerged. Here, we evaluate the impact of chemical space coverage and sample matrix on the accuracy of ionization efficiency-based quantification. We show that extending the chemical space of interest is crucial in improving the performance of quantification. Therefore, we extend the ionization efficiency-based quantification approach to hydroxylated PCBs in serum samples with a retraining approach that involves 14 OH-PCBs and validate it with an additional four OH-PCBs. The predicted and measured ionization efficiency values of the OH-PCBs agreed within the mean error of 2.1 × and enabled quantification with the mean error of 4.4 × or better. We observed that the error mostly arose from the ionization efficiency predictions and the impact of matrix effects was of less importance, varying from 37 to 165%. The results show that there is potential for predictive machine learning models for quantification even in very complex matrices such as serum. Further, retraining the already developed models provides a timely and cost-effective solution for extending the chemical space of the application area.