High-throughput Sample Preparation Research Articles

B-154 Development and Evaluation of an Automated High-Throughput Magnetic Bead-Based pre-treatment Platform for Lipid-Soluble Vitamins in Clinical Mass Spectrometry

Abstract Background Mass spectrometry has become a critical tool in clinical diagnostics due to its unparalleled sensitivity, specificity, and ability for multiplex analysis, redefining standards in small molecule detection. However, its efficacy is limited by complex sample preparation requirements, such as purification and enrichment. Traditional pre-treatment methods often involve labor-intensive, error-prone procedures that can dilute samples, thereby necessitating enhanced sensitivity in mass spectrometry. Addressing these challenges, we developed an automated, high-throughput pre-treatment platform using magnetic bead technology to streamline the preparation of small molecules for analysis via liquid chromatography-tandem mass spectrometry (LC-MS/MS). This study assesses the platform's effectiveness and reliability using lipid-soluble vitamins as a test case. Methods We developed a magnetic bead-based extraction process for lipid-soluble vitamins (Vitamins A, 25-hydroxyvitamin D2, 25-hydroxyvitamin D3, E, and K1) from clinical serum samples, utilizing Zybio's EXM3000 for automation. Protein-bound vitamins were dissociated using methanol and isopropanol, followed by magnetic bead extraction. The extracted vitamins underwent LC-MS/MS analysis. Comparative analysis with traditional liquid-liquid extraction methods, using 20 clinical samples and commercially available kits, evaluated this novel approach's analytical efficacy. Results The method showed excellent linearity (r > 0.99) for the target compounds. The imprecision ranged from 1.5% to 9.3%, and the accuracy (recovery rate) was between 91.5% and 113.4%. The limit of quantification, repeated six times, had a deviation of -8.5% to 13% and a CV of 1.5%-6.4%, meeting industry standards and EP document requirements. Comparisons with commercial kits in 20 clinical samples showed deviations within ±15%, and the 95% confidence interval for expected bias met the testing requirements. The method also demonstrated high correlation with the commercial kits, as evidenced by regression equations for Vitamin A: y = 0.9732x - 1.7985, 25-hydroxyvitamin D2: y = 1.0163x - 0.0055, 25-hydroxyvitamin D3: y = 1.0597x - 0.7968, Vitamin E: y = 1.0569x - 0.2917, and Vitamin K1: y = 1.0055x - 0.0039. Conclusions This study showed excellent performance of a novel automated high-throughput magnetic bead sample preparation platform for small molecules in clinical mass spectrometry. This technology overcomes the complexities associated with sample pre-treatment in clinical mass spectrometry and holds broad clinical application prospects in mass spectrometric analysis.

Intrinsic interlayer shear strength of graphite

Graphite holds significant values in the energy and electronics industries due to its unique properties. As a quintessential example of highly anisotropic materials, the shear strength measures one of its most fundamental mechanical properties. However, the lack of ideal materials and testing methods has led to a wide dispersion in the reported values. To address this issue, we utilized epitaxially grown single-crystal graphite and developed a high-throughput sample preparation method, along with a novel loading technique in this work. The intrinsic shear strength of AB-stacked graphite was determined to be τs = 62 MPa, by excluding the size effect in measurements. The results are further compared to highly oriented pyrolytic graphite specimens processed down to nanoscale thickness, highlighting the adverse impact of twisted single-crystalline interfaces between the graphitic layers. Additionally, we observed a distinctive failure mechanism with continuous and uniform cascade plastic slips across the thickness of graphite samples, which corresponds to an interlayer shear strength approaching τs. The intrinsic shear strength characterized in our work sets an upper limit for the interlayer shear resistance of graphite. The experimental procedure for measuring shear strength can be applied to other van der Waals materials.

A high-throughput sample preparation approach for the determination of nonsteroidal anti-inflammatory drugs in urine samples through membrane-based microextraction employing a green supramolecular solvent

Nonsteroidal anti-inflammatory drugs (NSAIDs) are a class of drugs with antipyretic, analgesic, and anti-inflammatory effects widely used worldwide. This study aimed at developing an environmentally friendly and high-throughput sample preparation method for the determination of six NSAIDs in urine samples followed by high-performance liquid chromatography coupled to diode-array detection (HPLC-DAD). The experimental workflow consisted of hollow-fiber microporous membrane liquid-liquid extraction (HF-MMLLE) using a supramolecular solvent immobilized into the porous of polypropylene hollow membranes (1 cm length). These membranes were attached to a 96-well plate system to permit multiple extractions simultaneously. Under the optimized conditions, limits of detection (LODs) and limits of quantification (LOQs) ranged from 6.06 to 30.3 µg/L and from 20 to 100 µg/L, respectively. The linear ranges varied from 20 to 1000 µg/L for indomethacin and diclofenac, from 50 to 1000 µg/L for ketoprofen and ibuprofen, and from 100 to 1000 µg/L for naproxen and fenoprofen. The coefficients of determination (R²) ranged from 0.9735 to 0.9997. Intraday precision ranged from 6.13 to 20.11 %, interday precision varied from 2.87 to 20.72 %, and accuracy ranged from 92.49 to 126.89 %. Moreover, the recently proposed Sample Preparation Metric of Sustainability (SPMS) was employed to evaluate the greenness of the sample preparation technique resulting in a score of 8.0, which is highly satisfactory according to the concepts of green analytical chemistry.

Exploring the Performance of Linear and Nonlinear Models of Time-of-Flight Secondary Ion Mass Spectrometry Spectra.

Multivariate statistical tools and machine learning (ML) techniques can deconvolute hyperspectral data and control the disparity between the number of samples and features in materials science. Nevertheless, the importance of generating sufficient high-quality sample replicates in training data cannot be overlooked, as it fundamentally affects the performance of ML models. Here, we present a quantitative analysis of time-of-flight secondary ion mass spectrometry (ToF-SIMS) spectra of a simple microarray system of two food dyes using partial least-squares (PLS, linear) and random forest (RF, nonlinear) algorithms. This microarray was generated by a high-throughput sample preparation and analysis workflow for fast and efficient acquisition of quality and reproducible spectra via ToF-SIMS. We drew insights from the bias-variance trade-off, investigated the performances of PLS and RF regression models as a function of training data size, and inferred the amount of data needed to construct accurate and reliable regression models. In addition, we found that the spectral concatenation of positive and negative ToF-SIMS spectra improved the model performances. This study provides an empirical basis for future design of high-throughput microarrays and multicomponent systems, for the purpose of analysis with ToF-SIMS and ML.

Functionalizing tandem mass tags for streamlining click-based quantitative chemoproteomics

Mapping the ligandability or potential druggability of all proteins in the human proteome is a central goal of mass spectrometry-based covalent chemoproteomics. Achieving this ambitious objective requires high throughput and high coverage sample preparation and liquid chromatography-tandem mass spectrometry analysis for hundreds to thousands of reactive compounds and chemical probes. Conducting chemoproteomic screens at this scale benefits from technical innovations that achieve increased sample throughput. Here we realize this vision by establishing the silane-based cleavable linkers for isotopically-labeled proteomics-tandem mass tag (sCIP-TMT) proteomic platform, which is distinguished by early sample pooling that increases sample preparation throughput. sCIP-TMT pairs a custom click-compatible sCIP capture reagent that is readily functionalized in high yield with commercially available TMT reagents. Synthesis and benchmarking of a 10-plex set of sCIP-TMT reveal a substantial decrease in sample preparation time together with high coverage and high accuracy quantification. By screening a focused set of four cysteine-reactive electrophiles, we demonstrate the utility of sCIP-TMT for chemoproteomic target hunting, identifying 789 total liganded cysteines. Distinguished by its compatibility with established enrichment and quantification protocols, we expect sCIP-TMT will readily translate to a wide range of covalent chemoproteomic applications.

Room-temperature fabrication of fluorinated covalent organic polymer @ Attapulgite composite for in-syringe membrane solid-phase extraction and analysis of domoic acid in aquatic products

A novel fluorinated covalent organic polymer @ attapulgite composite (F-COP@ATP) was prepared at room temperature for in-syringe membrane solid-phase extraction (SM-SPE) of domoic acid (DA) in aquatic products. Natural ore ATP has the advantages of low cost, good mechanical strength and abundant hydroxyl group on its surface, and in-situ modified F-COP layer can provide abundant adsorption sites. F-COP@ATP combining the advantages of F-COP and ATP, becomes an ideal adsorbent for DA extracting. Moreover, a high-throughput sample preparation strategy was carried out by using the F-COP@ATP membrane as syringe filter and assembling syringes with a ten-channel injection pump. In addition, the experimental factors were optimized, such as pH of extract, amount of adsorbent, velocity of extraction and desorption, type and volume of desorption solvent. The DA analytical method was established by SM-SPE-HPLC/tandem mass spectrometry. The method had a wide linear range with low limit of detection (0.344 ng/kg) and low limit of quantification (1.14 ng/kg). F-COP@ATP membrane can be reused more than five times. The method realized the analysis of DA in scallop and razor clam samples, which shows its application prospect in practical analysis. This study provided an efficient, low-energy and mild idea for preparing other reusable natural mineral ATP-based composite materials for separation and enrichment, which reduces the experimental cost and is closer to environmental protection and green chemistry to a certain extent.

Miniaturized on-spot protein denaturation/microwave-assisted extraction for spectrofluorimetric determination of favipiravir in dried plasma: Application to real human sample and inclusive incurred sample reanalysis study

The development of high-throughput sample preparation methods is a key step for upgrading the throughput of classical analytical techniques. In this work, a miniaturized on-spot protein denaturation coupled with microwave-assisted extraction sample treatment method was developed for extraction and subsequent spectrofluorimetric determination of favipiravir (FAV) in human plasma spots. Dried plasma spotting was performed using only 10 µL of plasma on filter paper discs followed by on-spot protein denaturation using 10 µL methanol. Maximum extraction efficiency was achieved using water: methanol (1:1) as extraction solvent with 50 % microwave power for 60 sec. The fluorescence intensity of extracted FAV was measured at 432 nm after excitation at 361 nm. According to the International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH -M10) guidelines for bioanalytical method validation, spiked plasma samples showed very good linearity between 100 and 1000 ng/mL. The accuracy and precision of the developed method were indicated by mean recovery (%) within 100 ± 15 % for all tested concentrations levels. The obtained results were further compared with a reported protein precipitation method using both F-test and t-test revealing no significant difference between either method. Moreover, the method was successfully applied for FAV determination in real human samples for an incurred sample reanalysis study revealing no metabolite back conversion. The developed method showed superior green performance using analytical Eco-scale, Green Analytical Procedure Index (GAPI), and Analytical Greenness Metric Approach (AGREE) tools compared to a classical protein precipitation method. According to Blue Applicability Grade Index (BAGI), and whiteness tools, the developed method outperformed all reported methods for FAV determination in plasma in terms of practicality and analytical functionality. Compared to reported sample treatment methods, this method exhibited excellent analytical performance, increased throughput, hundred-fold reduction of sample volume (10 μL), significant reduction of organic solvent consumption (260 μL methanol) and produced negligible wastes. Thus, it can be adopted for routine work in pharmacokinetics and clinical trial studies.

One step cleanup of 160 pesticides and veterinary drugs in aquatic products using melamine-based automatic pressure filtration purification method combined with HPLC-MS/MS

A 15-channel pressure filtration purification method was presented for high throughput sample preparation of aquatic products. A cost-effective device was constructed and melamine sponge was selected as the cleanup sorbent. Upon interfacing with HPLC-MS/MS, the analytical procedure demonstrated its suitability for quantifying 160 pesticides and veterinary drug residues in aquatic products such as fish, shrimp, and crab. The method achieved sample recoveries ranging from 61.3 to 124.9 %. The detection limits were established between 0.5 and 1.0 μg/kg, while the quantitation limits were confirmed to be within the range of 1.0–2.0 μg/kg. The method was applied to quantify the pesticide and veterinary drug residues in mostly consumed aquatic products from five coastal provinces in China. The results showed significant differences between different aquatic products in the concentrations of pesticide and veterinary drug residues, implying the necessity of supervision for the accurate determination of pesticides and veterinary drugs.

Single-Cell Proteomics Analysis with Tecan Uno and SCREEN Workflow.

With advances in sample preparation, small-volume liquid dispensing technologies, high-resolution MS/MS instrumentation, and data acquisition methodologies, it has become increasingly possible to confidently investigate the heterogeneous proteome found within individual cells. In this chapter, we present an automated high-throughput sample preparation workflow based on the Tecan Uno instrument for quantitative single-cell mass spectrometry-based proteomics. Cells are analyzed by the Single-Cell Proteome Analysis platform (SCREEN), which was introduced earlier and provides deeper proteome coverage across single cells.

Investigation of the sintering process and high-throughput sample preparation of Cu-Cr-Zr alloy

In this work, the influences of sintering temperature, duration, and pressure on the properties of Cu-0.6Cr-0.1Zr alloy were investigated by the spark plasma sintering through L9(33) orthogonal experiment design. The optimum parameters were determined at 850 °C with 50 MPa for 20 min. Subsequently, a high-throughput sample of Cu-xCr-0.1Zr (x = 0–0.9 wt%) was prepared under the aforementioned experimental conditions to explore the impact of Cr content on the microstructure and properties of Cu alloys. It was found that Cr addition enhanced mechanical properties while reducing the electrical conductivity in the alloy. By striking a balance between mechanical properties and electrical conductivity, an optimum Cr addition level of 0.6 wt% was identified for Cu-xCr-0.1Zr alloy.

Gelatin medium for preserving of Trichinella spp. for quality control in laboratories - estimation of larvae viability.

Official food control laboratories ensure food safety using reliable, validated methods. Council Regulations (EC) No. 853/2004, 854/2004 and 882/2004 of the European Parliament established hygiene rules the production of food of animal origin, together with requirements for official controls. This leads to detailed requirements for Trichinella control set out in Commission Implementing Regulation (EU) 2015/1375 of 10 August 2015. These regulations require the laboratory to participate in proficiency testing (PT) to confirm their competence and improve the quality of testing, and require the PT Organizer to use methods for the preparation and preservation of parasite larvae in order to evaluate and improve detection. Traditional methods of preparing such larvae expose them to rapid degradation, making it necessary to simultaneously isolate the larvae and place them in meatballs to ensure quality. We developed a technique for preserving of Trichinella spp. for quality control such as PT sample preparation. The procedure protects larvae against toxic oxygen activity and bacterial destruction via a gelatin barrier. To estimate the viability of larvae preserved by this method, gelatin capsules with 10 larvae of T. spiralis in each were stored (4-8 °C) during 45 days of an experiment. Samples were tested at 2 day intervals (3 samples each day of testing). In total, 75 samples were tested. Larvae remained alive up to 3 weeks. The number of living larvae diminished after 27 days through day 43, after which no living larvae were observed. The gelatin medium procedure facilitated easy, high-throughput sample preparation and supported 100% recovery for 3 weeks. The method allows fast, efficient and accurate PT sample preparation.

Mass-Activated Droplet Sorting for the Selection of Lysine-Producing Escherichia coli.

Synthetic biology relies on engineering cells to have desirable properties, such as the production of select chemicals. A bottleneck in engineering methods is often the need to screen and sort variant libraries for potential activity. Droplet microfluidics is a method for high-throughput sample preparation and analysis which has the potential to improve the engineering of cells, but a limitation has been the reliance on fluorescent analysis. Here, we show the ability to select cell variants grown in 20 nL droplets at 0.5 samples/s using mass-activated droplet sorting (MADS), a method for selecting droplets based on the signal intensity measured by electrospray ionization mass spectrometry (ESI-MS). Escherichia coli variants producing lysine were used to evaluate the applicability of MADS for synthetic biology. E. coli were shown to be effectively grown in droplets, and the lysine produced by these cells was detectable using ESI-MS. Sorting of lysine-producing cells based on the MS signal was shown, yielding 96-98% purity for high-producing variants in the selected pool. Using this technique, cells were recovered after screening, enabling downstream validation via phenotyping. The presented method is translatable to whole-cell engineering for biocatalyst production.

A high-throughput pipeline for DNA/RNA/small RNA purification from tissue samples for sequencing.

High-throughput total nucleic acid (TNA) purification methods based on solid-phase reversible immobilization (SPRI) beads produce TNA suitable for both genomic and transcriptomic applications. Even so, small RNA species, including miRNA, bind weakly to SPRI beads under standard TNA purification conditions, necessitating a separate workflow using column-based methods that are difficult to automate. Here, an SPRI-based high-throughput TNA purification protocol that recovers DNA, RNAand small RNA, calledGSC-modified RLT+ Aline bead-based protocol (GRAB-ALL), which incorporates modifications to enhance small RNA recoveryis presented. GRAB-ALL wasbenchmarkedagainst existing nucleic acid purification workflows and GRAB-ALL efficiently purifies TNA, including small RNA, for next-generation sequencing applications in a plate-based format suitable for automated high-throughput sample preparation.

Nail analysis for the assessment of caffeine abuse in Northwest China: A population-based study.

Caffeine is the most widely consumed psychoactive agent worldwide and has the potential for abuse, but studies monitoring caffeine abuse in China are scarce. This study aims to estimate the prevalence of caffeine abuse in northwest China and investigate the correlation between caffeine and other drugs in hair and nails using an ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). Fingernail clippings were collected from 376 participants in northwest China to detect caffeine and 13 other illicit psychoactive drugs and their metabolites. Paired hair and nail samples were collected from 39 participants to investigate the correlation between caffeine and other drugs in hair and nails. The samples were decontaminated, pulverized, and extracted by a high-throughput nail sample preparation method and analyzed by UPLC-MS/MS. The results showed a risk of caffeine abuse in northwest China, with concentrations ranging from 0.43 to 10.6 ng/mg for healthy volunteers, 0.49-246 ng/mg for caffeine abusers, and 0.25-363 ng/mg for drug addicts in community rehabilitation centers. Caffeine was detected together with other illicit psychoactive drugs and their metabolites. Furthermore, positive detection correlations were found between hair and nail samples. This study provides a current perspective on caffeine abuse in northwest China and demonstrates the practical use of UPLC-MS/MS for the simultaneous detection of caffeine and 13 illicit psychoactive drugs and their metabolites in hair and nails. The results highlight the potential of nails as a supplementary matrix when hair samples are unavailable and emphasize the need for handling caffeine carefully given its potential for abuse.

High-throughput quantification of microbial-derived organic acids in mucin-rich samples via reverse phase high performance liquid chromatography.

Organic acids (short chain fatty acids, amino acids, etc.) are common metabolic byproducts of commensal bacteria of the gut and oral cavity in addition to microbiota associated with chronic infections of the airways, skin, and soft tissues. A ubiquitous characteristic of these body sites in which mucus-rich secretions often accumulate in excess, is the presence of mucins; high molecular weight (HMW), glycosylated proteins that decorate the surfaces of non-keratinized epithelia. Owing to their size, mucins complicate quantification of microbial-derived metabolites as these large glycoproteins preclude use of 1D and 2D gel approaches and can obstruct analytical chromatography columns. Standard approaches for quantification of organic acids in mucin-rich samples typically rely on laborious extractions or outsourcing to laboratories specializing in targeted metabolomics. Here we report a high-throughput sample preparation process that reduces mucin abundance and an accompanying isocratic reverse phase high performance liquid chromatography (HPLC) method that enables quantification of microbial-derived organic acids. This approach allows for accurate quantification of compounds of interest (0.01 mM - 100 mM) with minimal sample preparation, a moderate HPLC method run time, and preservation of both guard and analytical column integrity. This approach paves the way for further analyses of microbial-derived metabolites in complex clinical samples.

A novel method of high-throughput micro-and nanomechanical sample fabrication.

There is an increasing interest in understanding materials' mechanical properties at small length scales. Mechanical testing from nano- to meso-scale has seen a rapid development over the last decade, leading to a high demand of sample fabrication. In the present work, a novel method of micro-and nanomechanical sample preparation is introduced based on a new technique combining femtosecond laser and focused ion beam (FIB), namely LaserFIB. The new method greatly simplifies the sample preparation workflow by taking advantage of the fast milling-rate of femtosecond laser and the high precision of FIB. It significantly improves the processing efficiency and success rate, allowing for the high-throughput preparation of reproducible micro- and nanomechanical specimens. The novel method has far more advantages: (1) it allows for site-specific sample preparation based on scanning electron microscope (SEM) characterization (lateral and depth direction of bulk material) (2) following the new workflow, mechanical specimens are still connected to the bulk by its natural bonding, yielding more reliable mechanical testing results; (3) it extends the processable sample size to meso-scale while still remaining high precision and high efficiency; (4) the seamless transfer between laser and FIB/SEM chamber greatly reduces the risk of sample damage and is very friendly for environmental sensitive materials. The new method solves critical problems for high-throughput multiscale mechanical sample preparation, greatly contributing to the development of nano to meso-scale mechanical testing by making sample preparation efficient and convenient.

An effective and high-throughput sample preparation method involving demalonylation followed by an ultrahigh-performance liquid chromatography–charged aerosol detector for analyzing gypenoside XLIX and gypenoside A in Gynostemma longipes

Gypenosides (Gps) are the major bioactive components in Gynostemma species. They include neutral Gps and acidic malonylgypenosides (MGps). MGps are abundant in Gynostemma species and can be transformed into corresponding Gps via extraction, concentration, and drying. If only the Gps were quantified and MGps were ignored, the quality of Gynostemma species would be underestimated. This study aimed to develop a sample preparation method involving demalonylation and ultrahigh-performance liquid chromatography–charged aerosol detector (UHPLC-CAD) analysis to determine the contents of gypenoside XLIX (Gp XLIX) and gypenoside A (Gp A). First, the optimized ultrasonic extraction method was established to extract G. longipes powder ultrasonically. Then, the extracted solution was put into a closed container (centrifuge tube) and heated in a water bath at 95 °C. Then, MGps were converted into corresponding Gps. The proposed preparation method was compared with the other three methods, including water bath reflux heating, alkali hydrolysis, and extraction of heated powder, and was shown to exhibit higher conversion and better convenience. Subsequently, an UHPLC-CAD method was established and validated. Gp XLIX and Gp A showed excellent linear correlations between 15.55 and 248.8 μg/mL and 24.10–385.5 μg/mL, respectively (R2 > 0.999). The limit of detection was 1.40 ng (Gp XLIX) and 2.41 ng (Gp A), and the limit of quantification was 7.77 ng and 14.46 ng, respectively. The relative standard deviation for precision, stability, and repeatability was 0.63–3.15%. The average recovery of Gp XLIX and Gp A was 98.97% and 98.23%, respectively. The established method was applied for determining Gp XLIX and Gp A contents in wild or cultivated G. longipes samples collected from the Qinba Mountains area. The contents of Gp XLIX and Gp A were 5.16–23.02 mg/g and 15.78–54.55 mg/g, respectively. Conclusively, the proposed sample preparation and analysis method could be used for the quality control and evaluation of G. longipes.

Coupling of Solid-Phase Microextraction Directly to Mass Spectrometry via an Improved Microfluidic Open Interface to Facilitate High-Throughput Determinations.

Mass spectrometry analysis can be performed by introducing samples directly to mass spectrometry, allowing the increase of the analysis throughput; however, some disadvantages of direct-to-mass spectrometry analysis include susceptibility to matrix effects and risk of instrument contamination from inadequate sample preparation. Solid-phase microextraction is one of the most suitable sample preparation methods for direct-to-mass spectrometry analysis, as it offers matrix-compatible coatings which ensure analyte enrichment with minimal or no interference from matrix. One of the ways solid-phase microextraction can be coupled directly to mass spectrometry is via a microfluidic open interface. This manuscript reports improvements made to the initial microfluidic open interface design, where the system components have been simplified to mostly commercially available materials. In addition, the analysis of samples has been automated by implementing software that fully controls the analysis workflow, where the washing procedure is optimized to completely reduce the carryover. Herein, the extraction and desorption time profiles from thin and thick SPME devices was studied where the overall workflow consisted of high-throughput sample preparation of 1.3 min per 96 samples and <1 min per sample instrumental analysis.

ASAP─Automated Sonication-Free Acid-Assisted Proteomes─from Cells and FFPE Tissues.

Formalin-fixed, paraffin-embedded (FFPE) tissues are an invaluable resource for retrospective studies, but protein extraction and subsequent sample processing steps have been shown to be challenging for mass spectrometry (MS) analysis. Streamlined high-throughput sample preparation workflows are essential for efficient peptide extraction from complex clinical specimens such as fresh frozen tissues or FFPE. Overall, proteome analysis has gained significant improvements in the instrumentation, acquisition methods, sample preparation workflows, and analysis pipelines, yet even the most recent FFPE workflows remain complex and are not readily scalable. Here, we present an optimized workflow for automated sonication-free acid-assisted proteome (ASAP) extraction from FFPE sections. ASAP enables efficient protein extraction from FFPE specimens, achieving similar proteome coverage as established methods using expensive sonicators, resulting in reduced sample processing time. The broad applicability of ASAP on archived pediatric tumor FFPE specimens resulted in high-quality data with increased proteome coverage and quantitative reproducibility. Our study demonstrates the practicality and superiority of the ASAP workflow as a streamlined, time- and cost-effective pipeline for high-throughput FFPE proteomics of clinical specimens.

Development of High-Throughput Sample Preparation Procedures for the Quantitative Determination of Aflatoxins in Biological Matrices of Chickens and Cattle Using UHPLC-MS/MS.

Aflatoxins (AFs) frequently contaminate food and animal feeds, especially in (sub) tropical countries. If animals consume contaminated feeds, AFs (mainly aflatoxin B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2) and their major metabolites aflatoxin M1 (AFM1) and M2 (AFM2)) can be transferred to edible tissues and products, such as eggs, liver and muscle tissue and milk, which ultimately can reach the human food chain. Currently, the European Union has established a maximum level for AFM1 in milk (0.05 µg kg-1). Dietary adsorbents, such as bentonite clay, have been used to reduce AFs exposure in animal husbandry and carry over to edible tissues and products. To investigate the efficacy of adding bentonite clay to animal diets in reducing the concentration of AFB1, AFB2, AFG1, AFG2, and the metabolites AFM1 and AFM2 in animal-derived foods (chicken muscle and liver, eggs, and cattle milk), chicken and cattle plasma and cattle ruminal fluid, a sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method has been developed. High-throughput sample preparation procedures were optimized, allowing the analysis of 96 samples per analytical batch and consisted of a liquid extraction using 1% formic acid in acetonitrile, followed by a further clean-up using QuEChERS (muscle tissue), QuEChERS in combination with Oasis® Ostro (liver tissue), Oasis® Ostro (egg, plasma), and Oasis® PRiME HLB (milk, ruminal fluid). The different procedures were validated in accordance with European guidelines. As a proof-of-concept, the final methods were used to successfully determine AFs concentrations in chicken and cattle samples collected during feeding trials for efficacy and safety evaluation of mycotoxin detoxifiers to protect against AFs as well as their carry-over to animal products.