Sample Preparation Time Research Articles

Immediate Diagnosis of Breast Carcinoma on Core Needle Biopsy Using Ex Vivo Fluorescence Confocal Microscopy: Feasibility in a One-Stop Breast Clinic Workflow

Background: In the one-stop breast clinic setting, breast cytology traditionally provides immediate diagnosis of carcinoma. Fluorescence confocal microscopy (FCM) is an emerging optical technique enabling ex vivo analysis of breast biopsies in real-time. This study represents the first proof of concept for integrating FCM imaging into the routine workflow of breast core needle biopsies (CNB) at Gustave Roussy’s one-stop breast clinic. Methods: Fifty women with breast masses underwent consecutive enrollment. Biopsies were stained with acridine orange and fast green, followed by imaging using the Vivascope 2500M-G4 (FCM). Interpretation was conducted by two pathologists in real time (PT1) or postoperatively (PT2). Concordance with definitive histology, the duration of the FCM protocol, and its impact on conventional histopathology, immunohistochemistry, and FISH analyses were evaluated. Results: In our study of 50 biopsies, a concordant diagnosis of malignancy was performed using FCM on the malignant cases at definitive histology in 93.5% (29/31 cases) and in 90.3% (28/31 cases) according to PT1 and PT2, respectively. When the FCM suspicious cases were added, FCM identified 100% (31/31 cases) and 96.7% (30/31 cases) of the malignant cases according to PT1 and PT2, respectively. A notable false positive case was identified as a complex sclerosing lesion. The median time for sample preparation (including tissue reception) was 5 min, while the median time for imaging acquisition with interpretation was 3 min for PT1, but 1 min required for interpretation alone by PT2. Histopathological alterations were not more prevalent in FCM-imaged biopsies compared to conventionally treated biopsies. The immunophenotyping and molecular assessment of tissue were preserved after FCM protocol. Conclusions: FCM shows promise as a new histological method for the immediate diagnosis of breast carcinoma on core needle biopsies in a one-stop clinic setting, while also preserving tissue specimens for final histology.

Development, Validation and Application of a Fast Sequential Method for Na, K, Ca and Mg Determination in Hemodialysis Solutions by HR-CS F AAS.

Hemodialysis solutions are liquid concentrates used during hemodialysis sessions. They are commonly used as a renal replacement therapy. This study aimed to develop a method to determine Na, K, Ca and Mg in hemodialysis solutions by high-resolution continuum source flame atomic absorption spectrometry (HR-CS F AAS) using Design of Experiments (DOE). The combination of the Doehlert Matrix with the desirability function was used to establish a compromise condition in the optimization of the significant variables studied: 60 L/h for acetylene flow rate, 8 mm for burner height, 0.25% (w/v) and 0.60% (w/v) for Cs and La concentrations. The method was validated by following the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) guidelines and parameters such as the selectivity, linearity, precision, accuracy and robustness were evaluated. Calibration curves with the ranges of 25-130 mg/L for Na, 1.0-5.0 mg/L for K and Ca, and 0.30-0.70 mg/L for Mg were employed. The method proved to be precise (RSD lower than 2.7%) and accurate (mean recovery data, contemplating the three levels added were: 103.0% for Na, 100.5% for K, 101.3% for Ca and 101.5% for Mg). The developed method enables the sequential multielement determination of Na, K, Ca and Mg in a single run. Requiring only one dilution step, this method significantly reduces analysis time for both sample and standard solutions preparation and measurement. This study presents the first method reported in the literature for multielement determination in hemodialysis solutions, offering an alternative approach to the current European Pharmacopeia method.

NanoMIP beacons with a co-operative binding mechanism for the all-in-one detection of methamphetamine aptamer complexes

Methamphetamine is a highly addictive stimulant with significant public health implications, necessitating the development of rapid, sensitive, and reliable detection methods. Traditional analytical techniques, though accurate, often involve complex sample preparation, expensive equipment, and lengthy analysis times. This study presents the design, synthesis, and application of nanoMIP beacons with a unique co-operative binding mechanism for the detection of methamphetamine. NanoMIP beacons selective for methamphetamine/aptamer complexes were synthesized using a solid-phase synthesis method that involved the bio-conjugation of an aptamer on the stationary phase and the incubation of methamphetamine to form a methamphetamine-aptamer complex. The resultant nanoMIP beacons were eluted off the affinity column and characterised using transmission electron microscopy. The co-operative binding mechanism, which relies on the structure-switching capability of the aptamer upon analyte binding was demonstrated through comparison of the fluorescence quenching signal of a scrambled sequence and nanoNIP controls. The fluorescence quenching assay was established using a fixed optimal concentration of aptamer and varying amounts of methamphetamine. The nanoMIP beacons showed enhanced the sensitivity (LOD = 23 ± 3.5 nM) and excellent selectivity, with a 40-fold increase in quenching for methamphetamine compared to other illicit drugs. The nanoMIP beacons demonstrated acceptable sample recoveries in both urine diluent and 50% human serum. This work provides a new strategy for the development of hybrid nanoMIP/aptamer-based sensors and provides a robust analytical tool for combating methamphetamine abuse.

3D Printed Coated Blade Spray-Mass Spectrometry Devices.

Coated blade spray-mass spectrometry (CBS-MS) has emerged as a powerful tool for the rapid screening of target compounds at trace levels in complex biological matrices. Despite its potential, the broader adoption of CBS-MS technology has been hindered by the lack of commercially available, user-friendly MS interfaces and extraction devices. In this work, we present comprehensive CBS-MS solutions developed using 3D printing, including a versatile MS interface and two extraction devices tailored to different analytical needs: one is compatible with LC vials for large-volume samples, while the other is optimized for single-drop blood analysis. The MS interface features a novel design that separates the immobilization station from the blade holder, significantly simplifying the operational workflow and minimizing the contamination risks and hazards associated with manual blade handling. The first extraction cartridge can process 48 samples simultaneously with an average sample preparation time of less than 20 s, while the second extraction device enables extraction from 8 single-drop blood samples via on-blade extraction. The developed devices were successfully tested for the rapid screening of seven drugs in both urine and single-drop blood samples, demonstrating a promising analytical performance. Additionally, potential contamination issues related to the use of 3D-printed materials as extraction phases were examined, emphasizing the importance of ensuring that 3D-printed materials do not leach contaminants into samples or solvents and contaminate the MS.

A-046 Quantification of Corticosteroids and Androgens in Serum, utilizing Waters MassTrak™ Steroid Serum Sets 2 & Sets 3 for Clinical Research

Abstract Background Steroid hormones encompass a large class of small molecules that play a central role in metabolic processes, such as the regulation of sexual characteristics, blood pressure, and inflammation. Enzymes that form part of the steroid biosynthetic pathway are pivotal in these metabolic processes, and their dysfunction can be examined through the correct measurement of steroid hormones in a clinical research setting. The availability of lyophilized calibrators and QCs reduces sample preparation time, aids in method harmonization, and assists with metrological traceability in accordance with ISO15189:2022, when used alongside an analytically selective chromatographic method. Methods A quantitative clinical research method utilizing Waters MassTrak Steroid Serum Sets 2 and 3, Cals & QCs and Waters Oasis™ PRiME HLB µElution plate technology for the extraction of testosterone, androstenedione, 17-hydroxyprogesterone (17- OHP), dehydroepiandrosterone sulfate (DHEAS), Cortisol, 11-deoxycortisol and 21-deoxycortisol from human serum samples. Chromatographic separation was performed on an Waters ACQUITY™ UPLC™ I Class Plus (FL-I) System, using an ACQUITY UPLC HSS T3 Column, accompanied by a Xevo™ TQ-S micro Mass Spectrometer. Results Accuracy (±6%) and precision (±10%) have been confirmed through comparison to External Quality Assurance (EQA) LC-MS/MS schemes, panels and QC material for all seven steroid hormones. All analytes were assessed at the low, medium and high concentrations for each MassTrak Steroid Serum Set, which yielded excellent results across the range (±10%). Deming and Linear regression analysis were performed. No statistically significant bias was observed for each compound, with a mean method bias of ±1.3% for Set 2 and ±1.0% for Set 3. The clinical research method was shown to be linear for all analytes over the calibration ranges specified, furthermore, calibration lines created using Set 2 and Set 3 were analysed over a five-day period and were linear with a co-efficient of determination of (r2) > 0.999 for all analytes. Analytical sensitivity using Signal:Noise (S/N) of the low calibrator (Calibrator 1) of each set, was >10:1 for each analyte across several analytical runs. Conclusions This evaluation has demonstrated that the MassTrak Steroid Serum Calibrator and Quality Control Sets 2 and 3 can provide precise and accurate quantification of steroid hormones in serum. An analytically sensitive and selective clinical research method has been developed for the analysis of testosterone, androstenedione, 17- OHP, DHEAS, cortisol, 11-deoxycortisol and 21-deoxycortisol in serum using Waters I-Class Xevo TQ-S micro system. For Research Use Only. Not for Use in Diagnostic Procedures.

B-248 Enhanced Detection of of Hemoglobin Variants in Clinical Research using Dried Blood Spot and HRAM MS

Abstract Background Hemoglobinopathies are one of the most prevalent genetic disorders, with more than 330,000 affected births occurring annually. Common techniques used in Hb analysis are electrophoretic and chromatographic assays. However, there are challenges to differentiate isomers (i.e., between Hb C and Hb E), very similar masses, or new variants due to their lack of resolution. Mass spectrometry (MS) gained popularity in clinical research because of its robustness and versatility in analyzing a wide range of samples and analytes from small molecules to proteins. Here, we present the top-down analysis for enhanced detection of various hemoglobin variants using Orbitrap Exploris for clinical research. Methods Proteins were directly extracted from dried blood spots (DBS) and analyzed by Thermo Scientific™ Orbitrap Exploris™ 240 with BioPharma Option coupled to Vanquish HPLC system. A 3.2 mm disc was punched from the DBS cards to 96 well plate and 50 uL water was added to re-hydrate. For protein precipitation, 150 uL acetonitrile was added and incubated at -20 ⁰C for 15 minutes. After micro-centrifugation, 160 uL of supernatant was removed and 70 uL water was added to resuspend. For LC-MS analysis, 10 uL of supernatant was diluted 8 times with mobile phase A and transferred to another 96-well plate. LC separation was performed on MAbPac RP column 4 um, 1 x 100 mm, and data-dependent acquisition mode was operated. Results With direct protein extraction from DBS, the entire sample preparation time is less than 1 hour. The MAbPac column showed its capability of separating between different chains with minor overlap between normal beta and HbS beta chains. Data processing was performed by ProSightPD, resulting in ∼45% sequence coverage for both normal Hb and HbS beta chains. The mass difference between normal beta and Hb S beta chains is about 30 Da, which results in about 1.6 m/z difference at a charge state of 19. This mass difference can often be hindered by other proteins or interferences from the matrix if the mass analyzer doesn’t have sufficient resolving power. Here we successfully applied Orbitrap resolution at 120,000 to isolate these similar masses for accurate variants or subunits identification including normal alpha, normal beta, Hb S, and delta chains. The confirmation of the delta chain can be beneficial since its abundance is usually less than 0.5% in adults, which shows potential for detecting low abundant variants using this method. Also, the calibration curve was generated to demonstrate the method's feasibility which is not a typical quantitation method for hemoglobinopathies. For quantitation, three most abundant m/z values from 2 charge states were added to TraceFinder for peak area extraction and normal Hb beta chain was used as an alternative internal standard for data processing. The 3-day inter/intraday analysis generated great reproducibility with CV < 10% and LOQ was determined to be 2.5 mg/mL with R2 values >0.994. Conclusions Successful demonstration of a single-step protein extraction directly from DBS and top-down analysis of Hb variant for clinical research.

Comparative study of bisphenols in e-cigarette liquids: evaluating fabric phase sorptive extraction, ultrasound-assisted membrane extraction, and solid phase extraction techniques

To address the under-researched risk of bisphenols (BPs) in e-cigarette liquids, comprehensive studies have been conducted to propose optimum sample preparation and analysis methods. To determine twelve BPs in refill liquids for e-cigarettes, three sample preparation methods based on distinct operational and working principles were employed. These included fabric phase sorptive extraction (FPSE), ultrasound-assisted solvent extraction of porous membrane-packed samples (UASE-PMS) and solid phase extraction (SPE) utilizing molecularly imprinted polymers (MIPs) technology. Each extraction method was combined with ultra-performance liquid chromatography coupled with tandem mass spectrometry (UPLS-MS/MS). Key parameters of FPSE and UASE-PMS procedures were optimized. This optimization included selection of the FPSE membrane types, durations of extraction and the choice solvents. Comprehensive validation was conducted, demonstrating linearity across a range from 2 to 60 ng/mL for all BPs (BPS, BPA, BPF, BPE, BPB, BPC, BPZ, BPFL, BPBP, BPP, BPG and BPM). Determination coefficients were above 0.9913, signifying linear relationship. The limits of detection (LODs) were established below 0.90 ng/mL, while the limits of quantification (LOQs) were lower than 2.5 ng/mL. Notably, the method based on UASE-PMS was successfully applied to the analysis of refill liquids for e-cigarettes samples. A comparative analysis of the methods highlighted variances in precision, accuracy, and applicable aspects, such as adjustment of parameters, sample preparation time, cost, handling, availability and possible limitations. Three methods have been identified as suitable for analysing BPs in e-cigarette refill liquids, highlighting the necessity to examine their presence in these products.

Programming-via-spinning: Electrospun shape memory polymer fibers with simultaneous fabrication and programming

Porous shape memory polymer (SMP) scaffolds are promising ‘smart’ materials for potential use in a wide range of biomedical applications. Electrospinning provides an approach to produce fibrous SMP scaffolds to enhance their porosity, mass transfer, and flexibility. Here, we studied the effects of electrospinning parameters (rotating collector rotational speed and solvent) on shape memory and mechanical properties of a biostable thermoplastic polyurethane (PUr) SMP. Scanning electron microscopy confirmed that fiber diameter and tortuosity could be tuned using varied collector rotation speeds and/or solvents. Mechanical properties, including modulus, tensile strength, and ultimate elongation, were tuned independently of chemistry based on variations in fiber architectures. All scaffolds demonstrated shape memory properties. Additionally, due to strains that are trapped in the fibers during the electrospinning process, SMP fibers are programmed into a strained, temporary shape during the fabrication step. These fibers can be immediately triggered to recover to a non-strained primary shape after fabrication to reduce sample preparation time and complexity. As a proof-of-concept, bacterial protease-responsive SMPs were electrospun and exposed to S. aureus in programmed secondary shapes. Upon exposure to bacteria, these SMPs underwent shape recovery, which resulted in reduced bacterial attachment and biofilm formation. These materials could be employed as bacteria-responsive wound dressings in future work. Overall, electrospinning provides a valuable tool for tuning mechanical and shape memory properties independently from chemistry and for programming SMPs during fabrication to enable scale-up of electrospun SMP scaffolds.

Direct analysis in real time mass spectrometry (DART-MS/MS) for rapid urine opioid detection in a clinical setting

Background and aimsCurrent laboratory methods for opioid detection involve an initial screening with immunoassays which offers efficient but non-specific results and a subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmation which offers accurate results but requires extensive sample preparation and turnaround time. Direct Analysis in Real Time (DART) tandem mass spectrometry is evaluated as an alternative approach for accurate opioid detection with efficient sample preparation and turnaround time. Materials and methodsDART-MS/MS was optimized by testing the method with varying temperatures, operation modes, extraction methods, hydrolysis times, and vortex times. The method was evaluated for 12 opioids by testing the analytical measurement range, percent carryover, precision studies, stability, and method-to-method comparison with LC-MS/MS. ResultsDART-MS/MS shows high sensitivity and specificity for the detection of 6-acetylmorphine, codeine, hydromorphone, oxymorphone, hydrocodone, naloxone, buprenorphine, norfentanyl, and fentanyl in urine samples. However, its performance was suboptimal for norbuprenorphine, morphine and oxycodone. ConclusionIn this proof-of-concept study, DART-MS/MS is evaluated for its rapid quantitative definitive testing of opioids drugs in urine. Further research is needed to expand its application to other areas of drug testing.

DiaPASEF Enables High-Throughput Proteomic Analysis of Host Cell Proteins for Biopharmaceutical Process Development.

Monitoring and quantifying host cell proteins (HCPs) in biotherapeutic production processes is crucial to ensure product quality, stability, and safety. Liquid chromatography-mass spectrometry (LC-MS) analysis has emerged as an important tool for identifying and quantifying individual HCPs. However, LC-MS-based approaches face challenges due to the wide dynamic range between HCPs and the therapeutic protein as well as laborious sample preparation and long instrument time. To address these limitations, we evaluated the application of parallel accumulation-serial fragmentation combined with data-independent acquisition (diaPASEF) to HCP analysis for biopharmaceutical process development applications. We evaluated different library generation strategies and LC methods, demonstrating the suitability of these workflows for various HCP analysis needs, such as in-depth characterization and high-throughput analysis of process intermediates. Remarkably, the diaPASEF approach enabled the quantification of hundreds of HCPs that were undetectable by a standard data-dependent acquisition mode while considerably improving sample requirement, throughput, coverage, quantitative precision, and data completeness.

Rapid detection of ricin at trace levels in complex matrices by asialofetuin-coated beads and bottom-up proteomics using high-resolution mass spectrometry

Ricin is a toxic protein regarded as a potential chemical weapon for bioterrorism or criminal use. In the event of a ricin incident, rapid analytical methods are essential for ricin confirmation in a diversity of matrices, from environmental to human or food samples. Mass spectrometry–based methods provide specific toxin identification but require prior enrichment by antibodies to reach trace-level detection in matrices. Here, we describe a novel assay using the glycoprotein asialofetuin as an alternative to antibodies for ricin enrichment, combined with the specific detection of signature peptides by high-resolution mass spectrometry. Additionally, optimizations made to the assay reduced the sample preparation time from 5 h to 80 min only. Method evaluation confirmed the detection of ricin at trace levels over a wide range of pH and in protein-rich samples, illustrating challenging matrices. This new method constitutes a relevant antibody-free solution for the fast and specific mass spectrometry detection of ricin in the situation of a suspected toxin incident, complementary to active ricin determination by adenine release assays.Graphical

A Bottom-Up Liquid Chromatography-Tandem Mass Spectrometry Method for Therapeutic Drug Monitoring of Infliximab: Method Development, Comparison With 2 Enzyme-Linked Immunosorbent Assay Methods, and Evaluation of Anti-Drug Antibody Interference.

Therapeutic drug monitoring is recommended to optimize infliximab use and improve outcome in chronic inflammatory disorders. To describe a simple and affordable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to measure infliximab in serum. Infliximab was measured using winged stable isotope-labeled peptides as internal standards. Linearity, lower limit of measuring interval, limit of detection, precision, accuracy, carryover, and ion suppression were evaluated. Method comparison against 2 enzyme-linked immunosorbent assay (ELISA) methods (Remsima Monitor and IDKmonitor Infliximab) and anti-drug antibody (ADA) interference were evaluated using clinical specimens from inflammatory bowel disease patients (N = 237). Analytical run time and sample preparation time were 5 minutes per sample and 3 hours per batch, respectively. Analytical measurement interval and limit of detection were 0.50 to 50.0 μg/mL (R2 = 0.998) and 0.25 μg/mL, respectively. The intraday and interday imprecision percentage coefficients of variation were less than 6.1%. Accuracy was 94.2% to 98.7%. No significant ion suppression or carryover was observed. Infliximab concentrations measured by LC-MS/MS showed good agreement with those measured by Remsima Monitor (mean percentage difference, 5.7%; 95% CI, -1.2% to 12.6%) but were markedly lower than those measured by IDKmonitor (-32.6%; -35.8% to -29.4%), demonstrating significant bias between ELISAs. Although a good agreement between LC-MS/MS and ELISA was observed for ADA-negative samples (-3.5%; -12.8% to 5.9%), a significant bias was observed for ADA-positive samples (13.6%; 1.7% to 25.6%). This simple, fast, and affordable LC-MS/MS method for infliximab quantitation could improve standardization of infliximab quantitation and optimization of infliximab use in patients with high-titer ADA.

Оптимізація пробопідготовки для визначення 90SR У харчових продуктах

The aim: To determine the possibility of conducting studies of the radionuclide 90Sr in food products in accordance with GN 6.6.1.1-130-2006 «Permissible levels of radionuclides 137Cs and 90Sr in food products and drinking wate»" without concentration by the ashing method. Materials and methods: The article describes the method of calculating the minimum measuring concentration of food products. According to the results of the calculations, the products that can be measured without concentration are determined. Analytical, statistical and computational research methods were used during the work. At the stage of sample preparation for measurement, the question often arises in which form to measure the sample - native or ashed. The specific activities of food products in their native form are in many cases lower than the minimum measurable concentration (MMС) on the spectrometer. Therefore, in order to decide in what form to prepare the sample for research, before starting work, it is necessary to compare the capabilities of the spectrometer, the minimum measured concentration and the permissible level of 90Sr content in the research product. In order to solve the question of the need for ashing of the sample, calculations of MMC were carried out for each food product, as well as the weight of the raw product, necessary to obtain 0.01 kg of ash per measurement was calculated. Results: Tables are compiled based on the results of the calculations. One includes products whose activity can be measured without ashing, and the other includes products with ashing. The use of such tables makes it possible to optimize sample preparation for the study of 90Sr by the spectrometric method. Of the 50 groups of food products, 17 have been identified that can be studied in their native form, which saves time for sample preparation and increases the performance of the spectrometer.

Determination of chlorothalonil in vegetables/fruits using liquid chromatography-tandem mass spectrometry with a discharge adapter interface.

Chlorothalonil (CHT), a broad-spectrum fungicide, has been employed widely to control foliar diseases, whereas with a major metabolite of polar 4-hydroxychlorothalonil (CHT-4-OH), only an acceptable nonpolar CHT residue is allowed by most countries. This study involves the method development for CHT residue in vegetables/fruits using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with a novel modified discharge-adaptor (DA) interface. CHT residue was analyzed using LC-MS/MS with DA interface (LC-DA-MS/MS), developed in our previous works. A DA was placed on the electrospray tip to switch the ionization modes. A modified quick, easy, cheap, effective, rugged, and safe (QuEChERS) method was applied to extract CHT residue of vegetables/fruits efficiently with less sample preparation time and analysis cost. CHT and CHT-4-OH spiked in four different vegetables/fruits were extracted using the modified QuEChERS method. After LC with isocratic elution, CHT and CHT-4-OH were separated within 3min. Using LC-DA-MS/MS, the ion signals of CHT were improved two to three times, and the limit of quantification of 5ng/g and linearity (r2 > 0.99) in the range of 5-200 ng/g were achieved using 10g of vegetables/fruits. The precision and accuracy were within 15% each. The modified QuEChERS and LC-DA-MS/MS were applied to examine eight field-grown vegetables/fruits; 9.5 and 2588.9ng/g of CHT were detected in two vegetables/fruits. LC-DA-MS/MS combined with modified QuEChERS was successfully applied to determine CHT residue <10ng/g in vegetables/fruits and with satisfied validation results. The developed method could reduce both analysis cost and time, attributing to simplifications in modified QuEChERS, isocratic elution, and DA interface in LC-DA-MS/MS.

Fermentation Analytical Technology (FAT): Monitoring industrial E. coli fermentations using absolute quantitative 1H NMR spectroscopy

BackgroundTo perform fast, reproducible, and absolute quantitative measurements in an automated manner has become of paramount importance when monitoring industrial processes, including fermentations. Due to its numerous advantages – including its inherent quantitative nature – Proton Nuclear Magnetic Resonance (1H NMR) spectroscopy provides an ideal tool for the time-resolved monitoring of fermentations. However, analytical conditions, including non-automated sample preparation and long relaxation times (T1) of some metabolites, can significantly lengthen the experimental time and make implementation in an industrial set up unfeasible. ResultsWe present a high throughput method based on Standard Operating Procedures (SOPs) and 1H NMR, which lays the foundation for what we call Fermentation Analytical Technology (FAT). Our method was developed for the accurate absolute quantification of metabolites produced during Escherichia coli industrial fermentations. The method includes: (1) a stopped flow system for non-invasive sample collection followed by sample quenching, (2) automatic robot-assisted sample preparation, (3) fast 1H NMR measurements, (4) metabolites quantification using multivariate curve resolution (MCR), and (5) metabolites absolute quantitation using a novel correction factor (k) to compensate for the short recycle delay (D1) employed in the 1H NMR measurements. The quantification performance was tested using two sample types: buffer solutions of chemical standards and real fermentation samples. Five metabolites – glucose, acetate, alanine, phenylalanine and betaine – were quantified. Absolute quantitation ranged between 0.64 and 3.40 mM in pure buffer, and 0.71–7.76 mM in real samples. SignificanceThe proposed method is generic and can be straight forward implemented to other types of fermentations, such as lactic acid, ethanol and acetic acid fermentations. It provides a high throughput automated solution for monitoring fermentation processes and for quality control through absolute quantification of key metabolites in fermentation broth. It can be easily implemented in an at-line industrial setting, facilitating the optimization of the manufacturing process towards higher yields and more efficient and sustainable use of resources.

An alternative method for predicting internal friction angle of rock materials

The shear strength properties of rock materials, cohesion and internal friction angle, are determined by carrying out tri-axial strength test on cylindrical core specimens in laboratory. But determination of these parameters by triaxial tests in accordance with standards and suggested methods, particularly for weak, fractured and weathered rocks is exteremely difficult and/or impossible due to difficulties related to preparation of test specimens suitable for this test. In addition, the tri-axial test requires high cost equipment and too much time for sample preparation and testing. In such cases, there is a need to precisely estimate the friction angle and estimation of rock shear strength properties using some indirect methods, as they are economical and easy to carry out. In this study, the traditional method, which is recommended to be used for the prediction of internal friction angle (ϕ) when triaxial test data is not available, was briefly assessed with its some limitations and an alternative method using theoretical tensile strength and uniaxial compressive strength to predict ϕ was proposed. Then the prediction performances of traditional and proposed methods were compared using a very large data set collected from published literature. The statistical reliability of the derived equations was assessed using F- and t-tests and according to the test results the prediction equations were found to be statistically reliable. The results indicated that the method proposed in this study using the theoretical tensile strength yields best predictions of ϕ when compared to those estimated from the traditional methods based on direct and Brazilian tensile strength values.

Distinguishing methicillin-resistant Staphylococcus aureus from methicillin-sensitive strains by combining Fe3O4 magnetic nanoparticle-based affinity mass spectrometry with a machine learning strategy

Pathogenic bacteria, including drug-resistant variants such as methicillin-resistant Staphylococcus aureus (MRSA), can cause severe infections in the human body. Early detection of MRSA is essential for clinical diagnosis and proper treatment, considering the distinct therapeutic strategies for methicillin-sensitive S. aureus (MSSA) and MRSA infections. However, the similarities between MRSA and MSSA properties present a challenge in promptly and accurately distinguishing between them. This work introduces an approach to differentiate MRSA from MSSA utilizing matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) in conjunction with a neural network-based classification model. Four distinct strains of S. aureus were utilized, comprising three MSSA strains and one MRSA strain. The classification accuracy of our model ranges from ~ 92 to ~ 97% for each strain. We used deep SHapley Additive exPlanations to reveal the unique feature peaks for each bacterial strain. Furthermore, Fe3O4 MNPs were used as affinity probes for sample enrichment to eliminate the overnight culture and reduce the time in sample preparation. The limit of detection of the MNP-based affinity approach toward S. aureus combined with our machine learning strategy was as low as ~ 8 × 103 CFU mL−1. The feasibility of using the current approach for the identification of S. aureus in juice samples was also demonstrated.Graphical

In-Membrane Enrichment and Peptic Digestion to Facilitate Analysis of Monoclonal Antibody Glycosylation.

The number of therapeutic monoclonal antibodies (mAbs) is growing rapidly due to their widespread use for treating various diseases and health conditions. Assessing the glycosylation profile of mAbs during production is essential to ensuring their safety and efficacy. This research aims to rapidly isolate and digest mAbs for liquid chromatography-tandem mass spectrometry (LC-MS/MS) identification of glycans and monitoring of glycosylation patterns, potentially during manufacturing. Immobilization of an Fc region-specific ligand, oFc20, in a porous membrane enables the enrichment of mAbs from cell culture supernatant and efficient elution with an acidic solution. Subsequent digestion of the mAb eluate occurred in a pepsin-modified membrane within 5 min. The procedure does not require alkylation and desalting, greatly shortening the sample preparation time. Subsequent LC-MS/MS analysis identified 11 major mAb N-glycan proteoforms and assessed the relative peak areas of the glycosylated peptides. This approach is suitable for the glycosylation profiling of various human IgG mAbs, including biosimilars and different IgG subclasses. The total time required for this workflow is less than 2 h, whereas the conventional enzymatic release and labeling of glycans can take much longer. Thus, the integrated membranes are suitable for facilitating the analysis of mAb glycosylation patterns.

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.

Isoporous Membrane Mediated Imprinting Mass Spectrometry Imaging for Spatially-Resolved Metabolomics and Rapid Histopathological Diagnosis.

Surface-assisted laser desorption/ionization (SALDI) mass spectrometry imaging (MSI) holds great value in spatial metabolomics and tumor diagnosis. Tissue imprinting on the SALDI target can avoid laser-induced tissue ablation and simplifies the sample preparation. However, the tissue imprinting process always causes lateral diffusion of biomolecules, thereby losing the fidelity of metabolite distribution on tissue. Herein, a membrane-mediated imprinting mass spectrometry imaging (MMI-MSI) strategy is proposed using isoporous nuclepore track-etched membrane as a mediating imprinting layer to selectively transport metabolites through uniform and vertical pores onto silicon nanowires (SiNWs) array. Compared with conventional direct imprinting technique, MMI-MSI can not only exclude the adsorption of large biomolecules but also avoid the lateral diffusion of metabolites. The whole time for MMI-based sample preparation can be reduced to 2min, and the lipid peak number can increase from 46 to 113 in kidney tissue detection. Meanwhile, higher resolution of MSI can be achieved due to the confinement effect of the pore channel in the diffusion of metabolites. Based on MMI-MSI, the tumor margins of liver cancer can be clearly discriminated and their different subtypes can be precisely classified. This work demonstrates MMI-MSI is a rapid, highly sensitive, robust and high-resolution technique for spatially-resolved metabolomics and pathological diagnosis.