High-throughput Quantitative Method Research Articles

A novel high-throughput quantitative method for the determination of per- and poly-fluoroalkyl substances in human plasma based on UHPLC-Q/Orbitrap HRMS coupled with isotope internal standard

A novel high-throughput quantitative method for the determination of per- and poly-fluoroalkyl substances in human plasma based on UHPLC-Q/Orbitrap HRMS coupled with isotope internal standard

Enhancing 2-Pyrone Synthase Efficiency by High-Throughput Mass-Spectrometric Quantification and In Vitro/In Vivo Catalytic Performance Correlation.

Engineering efficient biocatalysts is essential for metabolic engineering to produce valuable bioproducts from renewable resources. However, due to the complexity of cellular metabolic networks, it is challenging to translate success in vitro into high performance in cells. To meet such a challenge, an accurate and efficient quantification method is necessary to screen a large set of mutants from complex cell culture and a careful correlation between the catalysis parameters in vitro and performance in cells is required. In this study, we employed a mass-spectrometry based high-throughput quantitative method to screen new mutants of 2-pyrone synthase (2PS) for triacetic acid lactone (TAL) biosynthesis through directed evolution in E. coli. From the process, we discovered two mutants with the highest improvement (46 fold) in titer and the fastest kcat (44 fold) over the wild type 2PS, respectively, among those reported in the literature. A careful examination of the correlation between intracellular substrate concentration, Michaelis-Menten parameters and TAL titer for these two mutants reveals that a fast reaction rate under limiting intracellular substrate concentrations is important for in-cell biocatalysis. Such properties can be tuned by protein engineering and synthetic biology to adopt these engineered proteins for the maximum activities in different intracellular environments.

Optogenetic cytosol acidification of mammalian cells using an inward proton-pumping rhodopsin

Ion gradients are a universal form of energy, information storage and conversion in living cells. Advances in optogenetics inspire the development of novel tools towards control of different cellular processes with light. Rhodopsins are perspective tools for optogenetic manipulation of ion gradients in cells and subcellular compartments, controlling pH of the cytosol and intracellular organelles. The key step of the development of new optogenetic tools is evaluation of their efficiency. Here, we used a high-throughput quantitative method for comparing efficiency of proton-pumping rhodopsins in Escherichia coli cells. This approach allowed us to show that an inward proton pump xenorhodopsin from Nanosalina sp. (NsXeR) is a powerful tool for optogenetic control of pH of mammalian subcellular compartments. Further, we demonstrate that NsXeR can be used for fast optogenetic acidification of the cytosol of mammalian cells. This is the first evidence of optogenetic cytosol acidification by an inward proton pump at physiological pH values. Our approach offers unique opportunities to study cellular metabolism at normal and pathological conditions and might help to understand the role of pH dysregulation in cellular dysfunctions.

Development of an apolipoprotein E mimetic peptide–lipid conjugate for efficient brain delivery of liposomes

Liposomes are versatile carriers that can encapsulate various drugs; however, for delivery to the brain, they must be modified with a targeting ligand or other modifications to provide blood–brain barrier (BBB) permeability, while avoiding rapid clearance by reticuloendothelial systems through polyethylene glycol (PEG) modification. BBB-penetrating peptides act as brain-targeting ligands. In this study, to achieve efficient brain delivery of liposomes, we screened the functionality of eight BBB-penetrating peptides reported previously, based on high-throughput quantitative evaluation methods with in vitro BBB permeability evaluation system using Transwell, in situ brain perfusion system, and others. For apolipoprotein E mimetic tandem dimer peptide (ApoEdp), which showed the best brain-targeting and BBB permeability in the comparative evaluation of eight peptides, its lipid conjugate with serine–glycine (SG)5 spacer (ApoEdp-SG-lipid) was newly synthesized and ApoEdp-modified PEGylated liposomes were prepared. ApoEdp-modified PEGylated liposomes were effectively associated with human brain capillary endothelial cells via the ApoEdp sequence and permeated the membrane in an in vitro BBB model. Moreover, ApoEdp-modified PEGylated liposomes accumulated in the brain 3.9-fold higher than PEGylated liposomes in mice. In addition, the ability of ApoEdp-modified PEGylated liposomes to localize beyond the BBB into the brain parenchyma in mice was demonstrated via three-dimensional imaging with tissue clearing. These results suggest that ApoEdp-SG-lipid modification is an effective approach for endowing PEGylated liposomes with the brain-targeting ability and BBB permeability.

Quantitative analysis of fucosylated glycoproteins by immobilized lectin-affinity fluorescent labeling.

Human biofluids are often used to discover disease-specific glycosylation, since abnormal changes in protein glycosylation can discern physiopathological states. Highly glycosylated proteins in biofluids make it possible to identify disease signatures. Glycoproteomic studies on saliva glycoproteins showed that fucosylation was significantly increased during tumorigenesis and that glycoproteins became hyperfucosylated in lung metastases, and tumor stage is associated with fucosylation. Quantification of salivary fucosylation can be achieved by mass spectrometric analysis of fucosylated glycoproteins or fucosylated glycans; however, the use of mass spectrometry is non-trivial for clinical practice. Here, we developed a high-throughput quantitative method, lectin-affinity fluorescent labeling quantification (LAFLQ), to quantify fucosylated glycoproteins without relying on mass spectrometry. Lectins with a specific affinity for fucoses are immobilized on the resin and effectively capture fluorescently labeled fucosylated glycoproteins, which are further quantitatively characterized by fluorescence detection in a 96-well plate. Our results demonstrated that serum IgG can be accurately quantified by lectin and fluorescence detection. Quantification in saliva showed significantly higher fucosylation in lung cancer patients compared to healthy controls or other non-cancer diseases, suggesting that this method has the potential to quantify stage-related fucosylation in lung cancer saliva.

Rapid, sensitive, and high-throughput quantification of broad serological ceramides by using isotope dilution liquid chromatography-negative ion electrospray tandem mass spectrometry.

Ceramides are important intermediates in the metabolism of sphingolipids. High-throughput liquid chromatography-mass spectrometry has been used extensively for monitoring the levels of serological ceramides, but is still limited by inadequate coverage or lack of sensitivity. Herein, a rapid, sensitive, and high-throughput isotope dilution liquid chromatography-negative ion electrospray tandem mass spectrometry (IDLC-nESI-MS/MS) method was developed and verified for accurate quantification of 41 ceramides, involving ceramides with C16-20 sphingosine, dihydro-ceramide, and dehydro-ceramide. This method was validated with excellent linearity (R2 > 0.99) and good recovery in the range of 90-110%. Intra- and inter-day imprecision were below 5.57% and 7.83% respectively. The improved high-throughput quantitative method developed in this study may aid in the accurate characterization of ceramides for understanding ceramide biology and application in disease diagnosis.

Direct coupling of fiber-in-tube solid-phase microextraction with tandem mass spectrometry to determine amyloid beta peptides as biomarkers for Alzheimer's disease in cerebrospinal fluid samples

Current research efforts at neurological diseases have focused on identifying novel biomarkers to aid in diagnosis, to provide accurate prognostic information, and to monitor disease progression. This study presents the direct coupling of fiber-in-tube solid-phase microextraction to tandem mass spectrometry as a reliable method to determine amyloid beta peptides (Aβ38, Aβ40, and Aβ42) as biomarkers for Alzheimer's disease in cerebrospinal fluid (CSF) samples. To obtain the biocompatible fiber-in-tube SPME capillary, a PEEK tube segment was longitudinally packed with fine fibers [nitinol wires coated with a zwitterionic polymeric ionic liquid], to act as selective extraction medium. The fiber-in-tube SPME–MS/MS method integrated analyte extraction/enrichment and sample cleanup (exclusion of interferents) into one step. The method provided lower limits of quantification (LLOQ: 0.2 ng mL−1 for Aβ38 and 0.1 ng mL−1 for Aβ40 and Aβ42), high precision (CV lower than 11.6%), and high accuracy (relative standard deviation lower than 15.1%). This method was successfully applied to determine Aβ peptides in CSF samples obtained from AD patients (n = 8) and controls (healthy volunteers, n = 10). Results showed that Aβ42 levels in the CSF samples obtained from AD patients were significantly lower compared to healthy controls (p < 0.05). On the basis of the ROC analysis results, the Aβ42/Aβ40 ratio (AUC = 0.950, p < 0.01; 95%) performed significantly better than Aβ42 alone (AUC = 0.913, p < 0.01; 95%) in discriminating between AD patients and healthy controls and presented better diagnostic ability for AD. The novelties of this study are not only related to evaluating Aβ peptides as AD biomarkers, but also to demonstrating direct online coupling of fiber-in-tube SPME with MS/MS as a quantitative high-throughput method for bioanalysis.

A high-throughput quantitative method to evaluate peroxisome-chloroplast interactions in Arabidopsis thaliana.

Organelles contribute to plant growth via their movements and interactions, which ensure efficient metabolic flow and help plants adapt to environmental stress. Live-cell imaging of the interactions of organelles has been performed in yeast, plant, and animal cells. However, high-throughput quantitative methods are needed to simultaneously analyze the interactions of many organelles in living plant cells. Here, we developed a semi-automatic high-throughput method to quantitatively evaluate the interactions between peroxisomes and chloroplasts using a distance transformation algorithm and high-resolution 3D fluorescent images taken by confocal laser scanning microscopy. Using this method, we measured the 3D distance between the center of peroxisome and chloroplast surface in Arabidopsis thaliana. We then compared the distances between these organelles in leaf mesophyll cells under light and dark conditions. This distance was shorter in the light than in the dark, which is in agreement with the findings of previous studies. We used our method to evaluate peroxisome-chloroplast (plastid) interactions in different cell types in the light and dark, including guard, stem, and root cells. Like in mesophyll cells, the distance between the peroxisome and chloroplast was shorter in the light in guard and stem cells, but not in root cells, suggesting that photosynthetic plastids (chloroplasts) play important roles in these interactions. When leaf mesophyll cells were incubated under high-intensity light, the frequency of shorter distances between peroxisomes and chloroplasts significantly increased. Our high-throughput, semi-automatic method represents a powerful tool for evaluating peroxisome-chloroplast interactions in different types of plant cells under various environmental conditions.

Development of high-throughput UPLC-MS/MS using multiple reaction monitoring for quantitation of complex human milk oligosaccharides and application to large population survey of secretor status and Lewis blood group

Human milk oligosaccharides (HMOs) have attracted increasing attention due to the emerging evidence of their positive roles for infant’s health. A high-throughput method for absolute quantitation of the complex HMOs including multiple isomeric structures is important but very challenging, due to the highly divers nature and wide variation in content of HMOs from different individuals. Here we used UPLC-MS-MRM in the negative-ion mode for accurate quantitation of 23 complex HMOs in just 15 min. The selected oligosaccharides are in their native forms and include neutral and sialylated, fucosylated and non-fucosylated, linear and branched, and secretor and Lewis phenotype indicators. The well validated method with good sensitivity, recovery and reproducibility was then applied to a large population quantitative survey of 251 Chinese mothers from five different ethnic groups (Han, Zhuang, Hui, Mongolian and Tibetan) living in different geographical regions for their secretor’s status and Lewis phenotypes.

Simultaneous quantification of 70 elements in biofluids within 5 min using inductively coupled plasma mass spectrometry to reveal elementomic phenotypes of healthy Chinese adults

High-throughput quantification of the composition of all chemical elements (elementome) in biological samples is essential for understanding their diverse functions in large cohort studies. We, here, established an ICP-MS method to simultaneously quantify 70 elements in 50 μL biofluids within 5 min. This validated method had excellent quantification linearity (R2 > 0.998), sensitivity (with LOD as low as 1.0 ng/L), precision (CV<15%), accuracy (|RE|<20% except Hg), recovery (80–120%), throughput and coverage with minute samples. The method also showed good applicability to multiple biofluids including human serum, plasma, urine and goat serum samples. By using this method, we furture measured 70 elements in blood plasma samples from 758 Chinese adult participants and established the first reference intervals for the concentration of these elements from 127 healthy adults in this population. This offers a high-throughput quantitative elementomics method to define population elementomic phenotypes and for investigating the diverse biological functions of many elements in multiple biological matrices.

Quantitative Distribution Characterization and Correlation Study of Composition, Structure and Hardness of Rim Region in Railway Wheel.

The railway wheel is the key component of high-speed railway train. To assure the safety in service, higher requirements are put forward in this study for the composition, microstructure uniformity, and comprehensive properties of wheel materials. In this paper, the high throughput quantitative distribution characterization methods of composition, microstructure, inclusions and Vickers hardness of high-speed railway wheel materials based on the spark source original position analysis technique, high throughput scanning electron microscope (SEM) combined with image batch processing technology, and automatic two-dimensional quantitative distribution analysis technique of inclusions and micro hardness have been studied. The distribution trend of the content of nine elements, size and quantity of sulfides and oxides, ferrite area fraction, and Vickers hardness from the wheel tread surface to the radial depth of about 50 mm below the surface has been discussed. The influence of inclusions distribution on the element segregation and the effect of rim-chilling process with different water spraying angle on the distribution of microstructure and micro hardness have been investigated. It was found that unsynchronized cooling on both sides of the rim altered the phase behavior of ferrite and pearlite and obvious inhomogeneity distribution of ferrite appeared, which led to the asymmetrical Vickers hardness in areas near or away from the flange. Based on the quantitative characterization of area fraction and micro hardness on the same location of wheel rim, a statistical mapping relationship between ferrite area fraction and Vickers hardness was established.

Comprehensive quantitative method for neurotransmitters to study the activity of a sedative-hypnotic candidate using microdialysis and LC×LC-MS/MS

Neurotransmitters (NTs) and their metabolites play crucial roles in the regulation of the sleep-wake cycle. Thus, a comprehensive quantitative analysis of NTs would be useful in elucidating the potential mechanisms involved in sedative-hypnotic activities. In this study, we developed a high-throughput quantitative method based on a two-dimensional chromatography-mass spectrometry technique to simultaneously analyze 63 NTs and their metabolites in rat plasma, brain homogenate, and microdialysis samples from five different sleep-associated regions of the brain. Moreover, this method was used to study the neurochemical mechanism of an adenosine analog sedative-hypnotic candidate YZG-331. Most of the correlations between NTs were lost after the administration of the sedative, particularly in the caudate putamen (CPu) and dorsal raphe nucleus (DRN), indicating that the sleep-wake balance was affected. Administration of the adenosine analog YZG-331 could act similar as accumulation of adenosine, inducing adenosine and its metabolite adenine were decreased significantly in the CPu, accompanying with GABA, aspartate, and glutamate changed slightly by the communications between different neurons to further promote sleep. In addition, YZG-331 affected the metabolism of tryptophan and serotonin (5-HT) in the DRN and orbital frontal cortex (OFC). Melatonin and 5-hydroxyindole-3-acetic acid (a metabolite of 5-HT) were significantly increased in the OFC, and the levels of glutamate/glutamine, asparagine, and adrenaline were altered. Sleep homeostasis is a balance between the duration of sleep and wakefulness and is coordinated by all NTs. The high-throughput quantitative method introduced in this study may aid in revealing the temporal cohesion among NTs, evaluating sleep homeostasis, and determining the effects of sedative-hypnotic drugs.

High-Throughput Quantitation of Cannabinoids by Liquid Chromatography Triple-Quadrupole Mass Spectrometry.

The high-throughput quantitation of cannabinoids is important for the cannabis industry. As medicinal products increase, and research into compounds that have pharmacological benefits increase, and the need to quantitate more than just the main cannabinoids becomes more important. This study aims to provide a rapid, high-throughput method for cannabinoid quantitation using a liquid chromatography triple-quadrupole mass spectrometer (LC-QQQ-MS) with an ultraviolet diode array detector (UV-DAD) for 16 cannabinoids: CBDVA, CBDV, CBDA, CBGA, CBG, CBD, THCV, THCVA, CBN, CBNA, THC, Δ8-THC, CBL, CBC, THCA-A and CBCA. Linearity, limit of detection (LOD), limit of quantitation (LOQ), accuracy, precision, recovery and matrix effect were all evaluated. The validated method was used to determine the cannabinoid concentration of four different Cannabis sativa strains and a low THC strain, all of which have different cannabinoid profiles. All cannabinoids eluted within five minutes with a total analysis time of eight minutes, including column re-equilibration. This was twice as fast as published LC-QQQ-MS methods mentioned in the literature, whilst also covering a wide range of cannabinoid compounds.

Combined use of analyte protectants and precolumn backflushing for a robust, high-throughput quantitative determination of aroma compounds in cigarette mainstream smoke by gas chromatography-tandem mass spectrometry

Trace analysis of multi-targets in complex matrices such as cigarette smoke has always been a great challenge for analysts. In this paper, a high-throughput qualitative and quantitative method was developed and validated to determine 559 aroma compounds in mainstream smoke using gas chromatography-tandem mass spectrometry. Among four commonly used extraction solvents, dichloromethane showed obvious advantages in extracting the target aroma compounds with different polarity. An analyte protectant mixture consisting of 3-methoxy-1,2-propanediol, 1,2-octandiol, 1,2-nonanediol, and 1,2-tetradecanediol was first presented to compensate for the matrix effect, which was suitable for solvents of a wide polarity range such as hexane, dichloromethane, acetone, acetonitrile, and methanol. Moreover, the introduction of precolumn backflushing markedly improved the instrument robustness in analyzing complex cigarette smoke. The results showed that the system retained good stability after 30 days of injection. The proposed method performed well in terms of key validation parameters: accuracy (70.3–118.8% for 552 compounds), inter/intraday precision (0.1–10.7% and 0.2–18.2%), and sensitivity (limit of detection 0.12–102.54 ng/mL). Finally, this method was successfully applied to analyze 12 different commercial cigarette brands. The proposed method has good application prospects based on its robustness and may be used as a potential tool to transfer the cigarette design from an experience-dependent mode to a digital design mode. This method also shows potential in assaying aroma compounds in essentials, food or plant-based substrates.

High-throughput bioanalysis of sitagliptin in plasma using direct analysis in real time mass spectrometry and its application in the pharmacokinetic study thereof.

Sitagliptin is a dipeptidyl peptidase-IV inhibitor for the treatment of type 2 diabetes mellitus. In the present study, a sensitive and high-throughput quantitative method based on the direct analysis in real time tandem mass spectrometry has been developed and validated for the bioanalysis of sitagliptin in rat plasma without chromatographic separation. Sitagliptin and its internal standard retagliptin were detected in positive ion mode by multiple reaction monitoring transitions at m/z 408.2→235.0 and 465.2→260.1, respectively. The method includes a simple solid-phase extraction sample preparation procedure, through which appropriate and reproducible analytical results within the linear concentration range of 20-2000ng/mL have been achieved. The intra- and interday precisions were<10.6% and the accuracies were ranging from -8.17 to 2.60%. This method has been successfully applied to the pharmacokinetic study of sitagliptin after single intravenous administration in rats. This approach shows considerable promise of direct analysis in real time tandem mass spectrometry method in the high-throughput bioanalysis.

High-throughput and trace analysis of diazepam in plasma using DART-MS/MS and its pharmacokinetic application

A high-throughput quantitative analytical method based on Direct Analysis in Real Time tandem mass spectrometry (DART-MS/MS) has been developed and validated for the determination of diazepam in rat plasma, whereby analyzing of each sample needs merely 25 μL plasma, simple solid phase extraction sample preparation and 15 s acquisition time. The multiple reaction monitoring (MRM) transitions at m/z 285.2 → 193.1 and 316.0 → 270.0 were selected for the monitoring of diazepam and its internal standard clonazepam respectively. A good linearity within the range of 10–2000 ng/mL, an intra- and inter-day precisions within <7.78% as to an accuracy ranging from 1.04% to 7.92% have been achieved. The method has been successfully applied to the pharmacokinetic study of diazepam in rats’ plasma after a single intragastric administration at a dose of 10 mg/kg. The results indicate that this method fulfills the requirements of the bioanalysis in sensitivity and accuracy. It shows considerable promise for application of DART-MS to the quantitative investigation of other drugs.

Genetic screening reveals phospholipid metabolism as a key regulator of the biosynthesis of the redox-active lipid coenzyme Q

Mitochondrial energy production and function rely on optimal concentrations of the essential redox-active lipid, coenzyme Q (CoQ). CoQ deficiency results in mitochondrial dysfunction associated with increased mitochondrial oxidative stress and a range of pathologies. What drives CoQ deficiency in many of these pathologies is unknown, just as there currently is no effective therapeutic strategy to overcome CoQ deficiency in humans. To date, large-scale studies aimed at systematically interrogating endogenous systems that control CoQ biosynthesis and their potential utility to treat disease have not been carried out. Therefore, we developed a quantitative high-throughput method to determine CoQ concentrations in yeast cells. Applying this method to the Yeast Deletion Collection as a genome-wide screen, 30 genes not known previously to regulate cellular concentrations of CoQ were discovered. In combination with untargeted lipidomics and metabolomics, phosphatidylethanolamine N-methyltransferase (PEMT) deficiency was confirmed as a positive regulator of CoQ synthesis, the first identified to date. Mechanistically, PEMT deficiency alters mitochondrial concentrations of one-carbon metabolites, characterized by an increase in the S-adenosylmethionine to S-adenosylhomocysteine (SAM-to-SAH) ratio that reflects mitochondrial methylation capacity, drives CoQ synthesis, and is associated with a decrease in mitochondrial oxidative stress. The newly described regulatory pathway appears evolutionary conserved, as ablation of PEMT using antisense oligonucleotides increases mitochondrial CoQ in mouse-derived adipocytes that translates to improved glucose utilization by these cells, and protection of mice from high-fat diet-induced insulin resistance. Our studies reveal a previously unrecognized relationship between two spatially distinct lipid pathways with potential implications for the treatment of CoQ deficiencies, mitochondrial oxidative stress/dysfunction, and associated diseases.

Quantifying the Protein Levels of All Nuclear Hormone Receptors by Mass Spectrometry

Nuclear Receptors (NRs) are a family of ligand-activated transcription factors that control the expression of genes involved in a wide range of physiological processes. An atlas detailing the expression of all NRs at the mRNA level was completed in 2006 using quantitative PCR [Bookout et al. Cell 2006]. The comparative measurement of NRs at the protein level, however, has been hindered by the poor quality of commercially available antibodies, as well as the absence of a high throughput method for quantitation. To address this need, we are developing a mass spectrometry-based targeted proteomic assay to quantify the absolute amounts of NR protein in a panel of mouse tissues. NRs were overexpressed in HEK293 cells by transient transfection and protein was isolated. The cell lysates were digested with a combination of trypsin and Lys-C following the Multi-Enzyme Digestion Filter Aided Sample Preparation protocol. The peptides were desalted using an in-house made C18 tip, separated on an EASY-Spray C18 column (75 um x 50 cm, 3Å), and analyzed on a Thermo QExactive HF in Top20 data-dependent acquisition mode. Protein identifications were made using MaxQuant software, and the identifications were mined for members of the NR family. The NR peptides detected were searched against an in silico generated list of optimal NR peptides (filtered for uniqueness, length, absence of post translational modifications, and conservation between human and mouse). The matching peptides were validated by parallel reaction monitoring (PRM) and purchased as synthetic isotopes with a heavy terminal arginine or lysine. Peptide linearity, and lower limits of detection (LLOD) were estimated by spiking digests from a C57Bl/6 mouse liver lysate with increasing amounts of the labeled peptides and analyzing by PRM. Peptides that displayed non-linear behavior were excluded for quantitation. The LLOD were between 100 amol and 1.5 fmol on column. A test panel of tissues (cerebrum, hippocampus, cerebellum, liver, spleen, brown/white adipose, and kidney) showed that we could detect endogenous expression of NRs. To date, we have purchased and validated peptides for 44 of the 49 receptors. We used this assay to quantify the changes in NR protein expression in mouse livers in response to 16 hours of fasting. We found significant changes in the nuclear expression of CAR (3.1-fold increase), RXRβ (1.8-fold increase), SHP (3.9-fold decrease) and RARβ (2.0-fold decrease) in the fasted vs. fed state. Increased CAR activity with fasting was further supported by label-free quantitative proteomics on the same lysates which revealed 210 differentially expressed proteins (2-fold change, p<0.05), with 61 (29%) identified as known CAR target genes. Once complete, this assay will provide researchers with a robust quantitative tool to investigate changes in NR protein expression that will be widely applicable to endocrine research.

A Method for the Rapid Measurement of Alkylresorcinols in Flour, Bread and Related Products Based on 1H qNMR.

The main objectives of the current work were to investigate differences among flours from traditionally preserved Greek varieties of cereals, and especially those of wheat, and in parallel, to correlate those potential differences with the presence of bioactive natural ingredients. In this context, we developed a new, fast, and simple method for the measurement of total 5-alkylresorcinols in cereals and related foods by qNMR. Several types of flour (white or whole-grain) coming from wheat, i.e., Triticum dicoccum, T. monococcum, T. aestivum, T. durum and T. turgidum, corn, barley, rye and oat from a certified producer in Greece were used either as raw materials or for the production of bread, pasta and flakes. A small portion of the flour or the corresponding product was extracted with DMSO-d6. The liquid part was directly analyzed by NMR (400 MHz). The simplicity of the NMR spectrum of the total extract and the lack of overlapping peaks permitted the development of a high throughput quantitative method for the measurement of total bioactive alkylresorcinols in less than 15 min. Grains, whole grain flours and breads from old varieties of T. dicoccum and T.monococcum showed high contents of alkylresorcinols (455–1148 mg/Kg), while the same compounds were completely absent from white flour and the corresponding bread. The term high-phenolic flour is proposed to distinguish among flour types.

Development of a rapid reversed-phase liquid chromatographic method for total free thiol quantitation in protein therapeutics

Free thiols, or unpaired cysteines, are important product quality attributes in the therapeutic proteins due to their potential impact on the protein structure, bioactivity and stability. While many free thiol quantitation methods were developed for specific therapeutic formats, an unmet need still exists for a multiproduct, high-throughput method for free thiol quantitation. In this study, a workflow was established that combines N-cyclohexylmaleimide (NcHM) derivatization and high-throughput reversed-phase ultra-high performance liquid chromatography (RP-UHPLC) separation with superficially porous particle (SPP) column for quantitating total free thiols in monoclonal antibodies (mAbs), fragment antigen-binding (Fab), and bispecific antibodies (BsAbs). The NcHM derivatization increases the hydrophobicity of the free thiol variants and allows the further separation and quantitation with RP-UHPLC. A thorough evaluation of sample preparation, column selection, chromatographic condition and LC–MS peak identification was performed to optimize and characterize the method outputs. Method optimization resulted in a 42-minute total analysis time. Method qualification demonstrated suitable accuracy, precision, linearity, specificity and robustness. This high-throughput method is not only used for quantitation of total free thiols for both in-process testing and drug substance/drug product batch testing, but also for provide the positional distribution of the free thiols in the protein.