Stable Isotope-labeled Internal Standards Research Articles

Development of Noninvasive Method for the Automated Analysis of Nine Steroid Hormones in Human Saliva by Online Coupling of In-Tube Solid-Phase Microextraction with Liquid Chromatography–Tandem Mass Spectrometry

Accurate measurement of steroid hormones is crucial to elucidate new mechanisms of action and diagnose steroid metabolism-related diseases. This study presents a simple, sensitive, and automated analytical method for nine representative steroid hormones. The method involves on-line coupling of in-tube solid-phase microextraction (IT-SPME) with liquid chromatography–tandem mass spectrometry (LC–MS/MS). The steroid hormones were extracted and enriched on a Supel-Q PLOT capillary column using IT-SPME. Subsequently, they were separated and detected within 6 min using a Discovery HS F5-3 column and positive ion mode multiple reaction monitoring system via LC–MS/MS. Calibration curves of these compounds using each stable isotope-labeled internal standard (IS) showed linearity with correlation coefficients greater than 0.9990 in the range of 0.01–40 ng/mL, with limits of detection (S/N = 3) of 0.7–21 pg/mL. Moreover, intra- and inter-day variations were lower than 8.1 and 15% (n = 6), respectively. The recoveries of these compounds from saliva samples were in the range of 82–114%. The developed IT-SPME/LC–MS/MS method of steroid hormones is a highly sensitive, specific, and non-invasive analytical method that allows extraction and enrichment with no organic solvents, and enables direct automated online analysis by simply ultrafiltrating a small sample of saliva.

Miniature mass spectrometer-based point-of-care assay for quantification of metformin and sitagliptin in human blood and urine.

Metformin (MET) and sitagliptin (STG) are widely used as the first-line and long-term oral hypoglycemic agents for managing type 2 diabetes mellitus (T2DM). However, the current lack of convenient and rapid measurement methods poses a challenge for individualized management. This study developed a point-of-care (POC) assay method utilizing a miniature mass spectrometer, enabling rapid and accurate quantification of MET and STG concentrations in human blood and urine. By combining the miniature mass spectrometer with paper spray ionization, this method simplifies the process into three to four steps, requires minimal amounts of bodily fluids (50 μL of blood and 2 μL of urine), and is able to obtain quantification results within approximately 2min. Stable isotope-labeled internal standards were employed to enhance the accuracy and stability of measurement. The MS/MS responses exhibited good linear relationship with concentration, with relative standard deviations (RSDs) below 25%. It has the potential to provide immediate treatment feedback and decision support for patients and healthcare professionals in clinical practice.

Therapeutic Drug Monitoring of 6 New-Generation Antiseizure Medications Using a Mass Spectrometry Method: Analysis of 2-Year Experience in a Large Cohort of Korean Epilepsy Patients.

New-generation antiseizure medications (ASMs) are increasingly prescribed, and therapeutic drug monitoring (TDM) has been proposed to improve clinical outcome. However, clinical TDM data on new-generation ASMs are scarce. To develop and validate a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for therapeutic drug monitoring (TDM) of 6 new-generation ASMs in serum and analyze the clinical TDM data from a large cohort of Korean patients with epilepsy. Stable isotope-labeled internal standards were added to protein precipitations of serum. One microliter of sample was separated on Agilent Poroshell EC-C18 column, and lacosamide, perampanel, gabapentin, pregabalin, vigabatrin, and rufinamide were simultaneously quantified by Agilent 6460 triple-quad mass spectrometer in multiple-reaction monitoring mode. Linearity, sensitivity, precision, accuracy, specificity, carryover, extraction recovery, and matrix effect were evaluated. TDM data of 458 samples from 363 Korean epilepsy patients were analyzed. The method was linear with limit of detection less than 0.05 μg/mL in all analytes. Intraassay and interassay imprecisions were less than 5% coefficient of variation. Accuracy was within ±15% bias. Extraction recovery ranged from 85.9% to 98.8%. A total of 88% (403 of 458) were on polypharmacy, with 29% (118 of 403) using concomitant enzyme inducers. Only 38% (175 of 458) of the concentrations were therapeutic, with 53% (244 of 458) being subtherapeutic. Drug concentration and concentration-to-dose ratio were highly variable among individuals in all 6 ASMs. A simple and rapid LC-MS/MS method for TDM of 6 ASMs was developed and successfully applied to clinical practice. This large-scale TDM data could help establish an effective monitoring strategy for these drugs.

LC-MS/MS method for quick detection of vonoprazan fumarate in human plasma: Development, validation and its application to a bioequivalence study.

A liquid chromatography-tandem mass spectrometry method with vonoprazan fumarate-d4 as a stable isotope-labeled internal standard was developed and validated aiming at quantification of vonoprazan fumarate in human plasma for a bioequivalence study. Chromatographic separation was achieved by acetonitrile one-step protein precipitation using a gradient elution of 0.1% formic acid aqueous solution and acetonitrile with a run time of 3.65 min. Detection was carried out on a tandem mass spectrometer in multiple reaction monitoring mode via a positive electrospray ionization interface. The multiple reaction monitoring mode of precursor-product ion transitions for vonoprazan fumarate and vonoprazan fumarate-d4 were m/z 346.0 → 315.1 and 350.0 → 316.0, respectively. The linear range was 0.150-60.000 ng/ml. This method was fully validated with acceptable results in terms of selectivity, carryover, lower limit of quantification, calibration curve, accuracy, precision, dilution effect, matrix effect, stability, recovery and incurred sample reanalysis. A successful application of this method was realized in the bioequivalence study of vonoprazan fumarate tablet (20 mg) among healthy Chinese volunteers.

Use of Nonhuman Sera as a Highly Cost-Effective Internal Standard for Quantitation of Multiple Human Proteins Using Species-Specific Tryptic Peptides: Applicability in Clinical LC-MS Analyses.

Quantitation of proteins using liquid chromatography-tandem mass spectrometry (LC-MS/MS) is complex, with a multiplicity of options ranging from label-free techniques to chemically and metabolically labeling proteins. Increasingly, for clinically relevant analyses, stable isotope-labeled (SIL) internal standards (ISs) represent the "gold standard" for quantitation due to their similar physiochemical properties to the analyte, wide availability, and ability to multiplex to several peptides. However, the purchase of SIL-ISs is a resource-intensive step in terms of cost and time, particularly for screening putative biomarker panels of hundreds of proteins. We demonstrate an alternative strategy utilizing nonhuman sera as the IS for quantitation of multiple human proteins. We demonstrate the effectiveness of this strategy using two high abundance clinically relevant analytes, vitamin D binding protein [Gc globulin] (DBP) and albumin (ALB). We extend this to three putative risk markers for cardiovascular disease: plasma protease C1 inhibitor (SERPING1), annexin A1 (ANXA1), and protein kinase, DNA-activated catalytic subunit (PRKDC). The results show highly specific, reproducible, and linear measurement of the proteins of interest with comparable precision and accuracy to the gold standard SIL-IS technique. This approach may not be applicable to every protein, but for many proteins it can offer a cost-effective solution to LC-MS/MS protein quantitation.

Quantifying payloads of antibody‒drug conjugates using a postcolumn infused-internal standard strategy with LC‒MS

BackgroundAntibody‒drug conjugates (ADCs) are innovative biopharmaceutics consisting of a monoclonal antibody, linkers, and cytotoxic payloads. Monitoring circulating payload concentrations has the potential to identify ADC toxicity; however, accurate quantification faces challenges, including low plasma concentrations, severe matrix effects, and the absence of stable isotope-labeled internal standards (SIL-IS) for payloads and their derivatives. Previous studies used structural analogs as internal standards, but different retention times between structural analogs and target analytes may hinder effective matrix correction. Therefore, a more flexible approach is required for precise payload quantification. ResultsWe developed an LC‒MS/MS method incorporating a postcolumn-infused internal standard (PCI-IS) strategy for quantifying payloads and their derivatives of trastuzumab emtansine, trastuzumab deruxtecan, and sacituzumab govitecan, including DM1, MCC-DM1, DXd, SN-38, and SN-38G. Structural analogs (maytansine, Lys-MCC-DM1, and exatecan) were selected as PCI-IS candidates, and their accuracy performance was evaluated based on the percentage of samples within 80%–120% quantification accuracy. Compared to the approach without PCI-IS correction, exatecan enhanced the accuracy performance from 30-40%–100% for SN-38 and DXd, while maytansine and Lys-MCC-DM1 showed comparable accuracy for DM1 and MCC-DM1. This validated PCI-IS analytical method showed superior normalization of matrix effect in all analytes compared to the conventional internal standard approach. The clinical application of this approach showed pronounced differences in DXd and SN-38 concentrations before and after PCI-IS correction. Moreover, only DXd concentrations after PCI-IS correction were significantly higher in patients with thrombocytopenia (p = 0.037). SignificanceThis approach effectively addressed the issue of unavailability of SIL-IS for novel ADC payloads and provided more accurate quantification, potentially yielding more robust statistical outcomes for understanding the exposure-toxicity relationship in ADCs. It is anticipated that this PCI-IS strategy may be extrapolated to quantify payloads and derivatives in diverse ADCs, thereby providing invaluable insights into drug toxicity and fortifying patient safety in ADC usage.

A Simplified Proteomics LC-MRM-MS Assay for Determination of apoE Genotypes in Plasma Samples.

Apolipoprotein E (apoE), a polymorphic plasma protein, plays a pivotal role in lipid transportation. The human apoE gene possesses three major alleles (ε2, ε3, and ε4), which differ by single amino acid (cysteine to arginine) substitutions. The ε4 allele represents the primary genetic risk factor for Alzheimer's disease (AD), whereas the ε2 allele protects against the disease. Knowledge of a patient's apoE genotype has high diagnostic value. A recent study has introduced an LC-MRM-MS-based proteomic approach for apoE isoform genotyping using stable isotope-labeled peptide internal standards (SIS). Here, our goal was to develop a simplified LC-MRM-MS assay for identifying apoE genotypes in plasma samples, eliminating the need for the use of SIS peptides. To determine the apoE genotypes, we monitored the chromatographic peak area ratios of isoform-specific peptides relative to a peptide that is common to all apoE isoforms. The assay results correlated well with the standard TaqMan allelic discrimination assay, and we observed a concordance between the two methods for all but three out of 172 samples. DNA sequencing of these three samples has confirmed that the results of the LC-MRM-MS method were correct. Thus, our simplified UPLC-MRM-MS assay is a feasible and reliable method for identifying apoE genotypes without using SIS internal standard peptides. The approach can be seamlessly incorporated into existing quantitative proteomics assays and kits, providing additional valuable apoE genotype information. The principle of using signal ratios of the protein isoform-specific peptides to the peptide common for all of the protein isoforms has the potential to be used for protein isoform determination in general.

Mitigating matrix effects for LC-MS/MS quantification of nitrosamine impurities in rifampin and rifapentine

Since the discovery of potentially mutagenic nitrosamine impurities in pharmaceuticals in 2018, liquid chromatography-mass spectrometry (LC-MS) methods have been widely applied for the trace level quantification of nitrosamines. As more nitrosamines and drug samples are analyzed, method accuracy is increasingly recognized as a major analytical challenge. Matrix effects can have a significant adverse impact on method accuracy but have not attracted much attention in nitrosamine quantification literature. In this study, we observed severe matrix effects when adapting LC-MS method conditions from a published and validated method for the analysis of nitrosamines in tuberculosis drugs. Several approaches were explored to mitigate these matrix effects, including using stable isotope-labeled internal standards, modifying LC conditions and sample concentration, and quantifying with standard addition instead of an external calibration method. The results underscore the importance of monitoring matrix effects and validating nitrosamine quantification procedures for specific sample matrices. Finally, two LC-MS/MS methods are presented with validation data for the quantification of 1-methyl-4-nitrosopiperazine (MNP) in rifampin and 1-cyclopentyl-4-nitrosopiperazine (CPNP) in rifapentine and revealed high nitrosamine levels in the tested samples that far exceeded recommended acceptable intake limits.

Investigation of variability in the matrix effect on stable isotope-labeled internal standards in liquid chromatography-tandem mass spectrometry analysis of 25 pesticides in vegetables

Investigation of variability in the matrix effect on stable isotope-labeled internal standards in liquid chromatography-tandem mass spectrometry analysis of 25 pesticides in vegetables

Probing Liquid Chromatography-Tandem Mass Spectrometry Response Dynamics and Nonlinear Effects for Response Level Defined Calibration Strategies with Simple Methods To Expand Linear Dynamic Ranges.

The response characteristics of liquid chromatography-tandem mass spectrometry (LC-MS/MS) serve as the basis for selecting calibration methods in quantitative analysis. LC-MS/MS inherently exhibits nonlinear detection behavior, primarily attributed to the disproportionate growth observed between peak area and peak height at elevated response levels, potentially leading to signal saturation. This disproportionate peak growth results in reduced unit response (UR), which quantifies the instrument's detection sensitivity. LC-MS/MS typically operates within a narrow near-linear response range (NLRR) due to approximately proportional peak growth, yet the NLRR width varies across different analytes or platforms. Although the inclusion of stable isotope-labeled (SIL) internal standards (IS) in LC-MS/MS analysis can mitigate certain instrument response variations, it does not eliminate the fundamental cause of nonlinearity. Moreover, the concentration range accommodated by the NLRR can significantly fluctuate at different sensitivity levels. LC-MS/MS also encounters various other nonlinear effects, including ion suppression during ionization, signal cross-contribution between the analyte/IS, and matrix effects (ME). Consequently, quadratic regression emerges as a more adaptable approach to LC-MS/MS nonlinear response dynamics, offering a broader calibration range. The application of linear regression, on the other hand, requires strict conditions. Although the signal saturation zone typically remains inaccessible to calibration methods, reducing responses by employing less-optimal selected reaction monitoring (SRM) transitions and/or lower detection gain can facilitate fitting a wide concentration range into the NLRR, thereby enabling accurate linear regression calibration. This report delves into examining the LC-MS/MS response profile, its dynamics, and major nonlinear effects through instrument response mapping to elucidate their influence on the selection of calibration methods.

Multiplexed Antibody Glycosylation Profiling Using Dual Enzyme Digestion and Liquid Chromatography-Triple Quadrupole Mass Spectrometry Method

Antibody glycosylation plays a crucial role in the humoral immune response by regulating effector functions and influencing the binding affinity to immune cell receptors. Previous studies have focused mainly on the immunoglobulin G (IgG) isotype owing to the analytical challenges associated with other isotypes. Thus, the development of a sensitive and accurate analytical platform is necessary to characterize antibody glycosylation across multiple isotypes. In this study, we have developed an analytical workflow using antibody-light-chain affinity beads to purify IgG, IgA, and IgM from 16 μL of human plasma. Dual enzymes, trypsin and Glu-C, were used during on-bead digestion to obtain enzymatic glycopeptides and protein-specific surrogate peptides. Ultra-high-performance liquid chromatography coupled with triple quadrupole mass spectrometry was used in order to determine the sensitivity and specificity. Our platform targets 95 glycopeptides across the IgG, IgA, and IgM isotypes, as well as eight surrogate peptides representing total IgG, four IgG classes, two IgA classes, and IgM. Four stable isotope-labeled internal standards were added after antibody purification to calibrate the preparation and instrumental bias during analysis. Calibration curves constructed using serially diluted plasma samples showed good curve fitting (R2 > 0.959). The intrabatch and interbatch precision for all the targets had relative standard deviation of less than 29.6%. This method was applied to 19 human plasma samples, and the glycosylation percentages were calculated, which were comparable to those reported in the literature. The developed method is sensitive and accurate for Ig glycosylation profiling. It can be used in clinical investigations, particularly for detailed humoral immune profiling.

Quantitative LC-MS/MS Analysis of Amphetamines and Cocaine in Human Urine.

We hereby present a fast and high-throughput LC-MS/MS assay for the simultaneous analysis of amphetamines and cocaine in human urine. The assay is used for confirmations following immunoassay urine drug screens as well as a quantitative assay to report actual urine concentrations in the range 30-10,000ng/mL for each of the seven analytes, namely, amphetamine; methamphetamine; phentermine; methylenedioxyamphetamine; 3,4-methylenedioxymethamphetamine; methylenedioxy-ethyl-amphetamine; and a cocaine metabolite, benzoylecgonine. The assay derives its efficacy from minimal sample preparation via dilute and shoot. The platform is based on reversed-phase liquid chromatography coupled to the TSQ Endura triple-quadrupole (QqQ) MS instrument for detection via electrospray ionization multiple-reaction monitoring MS. The quantitative analysis is based on the linear calibration whereby the instrument response for each analyte at a given concentration is normalized against stable isotope-labeled internal standard. In addition, the assay can be multiplexed across more than one LC channel to obtain high-sample throughput.

Associations of urinary caffeine and caffeine metabolites with metabolic syndrome in US adults.

The relationship between caffeine and metabolic syndrome (MetS) has only been evaluated from the perspective of caffeine consumption. The association between urinary caffeine and MetS is still unclear. This study examined the associations between urinary caffeine and its metabolites and MetS and its components among adults. Data from the United States (US) National Health and Nutrition Examination Survey (NHANES) 2011-2014 was analyzed. NHANES is a stratified, multi-stage survey of all non-institutionalized persons in the US. A total of 2,394 subjects aged ≥ 18 years without missing data were selected in this study. Urinary caffeine and caffeine metabolite levels were quantified using high-performance liquid chromatography-electrospray ionization-tandem quadrupole mass spectrometry (HPLC-ESI-MS/MS) with stable isotope-labeled internal standards. We performed principal components analysis (PCA) to investigate the underlying correlation structure of 15 features of urinary caffeine and its metabolites and then used these principal components (PCs) as independent variables to conduct logistic regression analysis with or without restricted cubic spline (RCS) terms to explore the associations between caffeine metabolites and MetS. Two main PCs that were derived from the PCA explained 90.67% of the total variance of caffeine and its metabolites. The first PC (PC1, strongly correlated with 1-MU, 1,3-DMU, 1,7-DMU, 1,3,7-TMU, 1-MX, 1,3-DMX, 1,7-DMX, 1,3,7-TMX, and AAMU) was positively correlated with risk of MetS (OR = 1.27, p < 0.001) and all its components (all ORs > 1, all p-values < 0.001) in the unadjusted models, while in the adjusted models, it was positively correlated with MetS (OR = 1.16, p = 0.042) and central obesity (OR = 1.22, p < 0.001). In the unadjusted model, there were significant associations between the second PC (PC2, correlated with 3-MU, 7-MU, 3,7-DMU, 3-MX, 7-MX, and 3,7-DMX) and MetS (OR = 1.11, P = 0.030) and central obesity (OR = 1.16, P < 0.001), while in the adjusted models (adjustment variables include gender, age, race/ethnicity, education level and income-poverty ratio, smoking status, drinking, and physical activity), PC2 was positively associated with MetS (OR = 1.15, p = 0.035) and central obesity (OR = 1.15, p = 0.005) and negatively associated with raised triglycerides (TG) (OR = 0.84, p = 0.008). Moreover, we observed U-shaped associations between PC1 and the risk of raised TG both in unadjusted (Pnon-linear = 0.017) and adjusted (Pnon-linear = 0.014) models. Urinary caffeine metabolites were positively associated with the risk of MetS and its components through different linear or non-linear patterns.

High-Throughput Comprehensive Quantitative LC-MS/MS Analysis of Common Drugs and Metabolites (62 Compounds) in Human Urine.

In recent years a multitude of LC-MS/MS assays have been widely reported in commercial and clinical literature demonstrating the simultaneous analyses of dozens of drugs of abuse in human samples. The utility of such assays is meant to supplant the indirect detection based on the classical spectral library approach. Direct and simultaneous analysis via LC-MS/MS technology is made possible by fast acquisition rates in multiple reaction monitoring, as well as sensitivity and high selectivity of the technology for each individual analyte in a complex mixture. Hence, unlike immunoassays, which are not well-suited for the analyses of mixtures, and which may also be prone to false positives from potential interferences, quantitative LC-MS/MS analyses are feasible for complex patient mixtures of drugs of abuse. We hereby present a robust clinical LC-MS/MS assay for the simultaneous and semi-quantitative analysis of up to 62 drugs of abuse in human urine, representing major classes that include opiates, benzodiazepines, amphetamines, etc. The assay utilizes dilute and shoot, whereby the sample is diluted ten times in internal standard reagent and thereafter submitted to the LC-MS instrument, i.e., reversed-phase liquid chromatography coupled to the electrospray ionization multiple reaction monitoring analysis, via the TSQ Endura triple-quadrupole instrument. The assay employs stable isotope-labeled internal standards with a linear response in the 30-300ng/mL range, effectively semi-quantitative, since this analytical range is well within typical immunoassay cutoffs for most drugs.

Comparison of colorimetric, fluorometric, and liquid chromatography-mass spectrometry assays for acetyl-coenzyme A

Acetyl-Coenzyme A is a central metabolite in catabolic and anabolic pathways as well as the acyl donor for acetylation reactions. Multiple quantitative measurement techniques for acetyl-CoA have been reported, including commercially available kits. Comparisons between techniques for acetyl-CoA measurement have not been reported. This lack of comparability between assays makes context-specific assay selection and interpretation of results reporting changes in acetyl-CoA metabolism difficult. We compared commercially available colorimetric ELISA and fluorometric enzymatic-based kits to liquid chromatography-mass spectrometry-based assays using tandem mass spectrometry (LC-MS/MS) and high-resolution mass spectrometry (LC-HRMS). The colorimetric ELISA kit did not produce interpretable results even with commercially available pure standards. The fluorometric enzymatic kit produced comparable results to the LC-MS-based assays depending on matrix and extraction. LC-MS/MS and LC-HRMS assays produced well-aligned results, especially when incorporating stable isotope-labeled internal standards. In addition, we demonstrated the multiplexing capability of the LC-HRMS assay by measuring a suite of short-chain acyl-CoAs in a variety of acute myeloid leukemia cell lines and patient cells.

A Single-Run HPLC-MS Multiplex Assay for Therapeutic Drug Monitoring of Relevant First- and Second-Line Antibiotics in the Treatment of Drug-Resistant Tuberculosis.

The treatment of drug-resistant Mycobacterium tuberculosis relies on complex antibiotic therapy. Inadequate antibiotic exposure can lead to treatment failure, acquired drug resistance, and an increased risk of adverse events. Therapeutic drug monitoring (TDM) can be used to optimize the antibiotic exposure. Therefore, we aimed to develop a single-run multiplex assay using high-performance liquid chromatography-mass spectrometry (HPLC-MS) for TDM of patients with multidrug-resistant, pre-extensively drug-resistant and extensively drug-resistant tuberculosis. A target profile for sufficient performance, based on the intended clinical application, was established and the assay was developed accordingly. Antibiotics were analyzed on a zwitterionic hydrophilic interaction liquid chromatography column and a triple quadrupole mass spectrometer using stable isotope-labeled internal standards. The assay was sufficiently sensitive to monitor drug concentrations over five half-lives for rifampicin, rifabutin, levofloxacin, moxifloxacin, bedaquiline, linezolid, clofazimine, terizidone/cycloserine, ethambutol, delamanid, pyrazinamide, meropenem, prothionamide, and para-amino salicylic acid (PAS). Accuracy and precision were sufficient to support clinical decision making (≤±15% in clinical samples and ±20-25% in spiked samples, with 80% of future measured concentrations predicted to fall within ±40% of nominal concentrations). The method was applied in the TDM of two patients with complex drug-resistant tuberculosis. All relevant antibiotics from their regimens could be quantified and high-dose therapy was initiated, followed by microbiological conversion. In conclusion, we developed a multiplex assay that enables TDM of the relevant first- and second-line anti-tuberculosis medicines in a single run and was able to show its applicability in TDM of two drug-resistant tuberculosis patients.

Simultaneous quantification of seven B vitamins in human faeces by stable isotope label-based high-performance liquid chromatography-tandem mass spectrometry

B vitamins in the human distal gut are primarily derived from the gut microbiota because daily dietary vitamins are fully absorbed in the small intestine under normal dietary and physiological conditions. Quantitative determination of B vitamins in the distal gut and faecal samples is crucial for understanding the intricate relationship between gut B vitamins, gut microbiota, and host health. In this study, we developed a rapid, robust, and reliable method with a simple extraction procedure for the simultaneous analysis of seven B vitamins in human faeces using high-performance liquid chromatography-electrospray ionisation-tandem mass spectrometry (HPLC-ESI-MS/MS) with stable isotope-labelled internal standards. A protein precipitation approach using methanol as the precipitant was employed to extract vitamin B from human faecal samples. Seven B vitamins were adequately separated and quantified within 9 min by HPLC-ESI-MS/MS with a Pursuit PFP column (2.0 ×150 mm, 3.0 µm), including vitamins B1, B2, B3, B5, pyridoxic acid, pyridoxine, and B7. The lower limits of quantification were within the range of 0.1–25 ng mL−1. The intra-day and inter-day precision and accuracy were both within 15 %. The validated method was successfully applied to 55 faecal samples collected from healthy individuals, patients with type 2 diabetes, and obese patients. Compared with healthy controls, obese patients had lower faecal concentrations of vitamins B1 and B3 and pyridoxic acid, whereas patients with type 2 diabetes had lower faecal concentrations of vitamins B1 and B5.

Synthesis of S-Carbamidomethyl Cysteine and Its Use for Quantification of Cysteinyl Peptides by Targeted Proteomics.

Proteomics has played a central role in the identification of reliable disease biomarkers, which are the basis of precision medicine, a promising approach for tackling recalcitrant diseases such as cancer, that elude conventional treatments. Among proteomic methodologies, targeted proteomics employing stable isotope-labeled (SIL) internal standards is particularly suited for the clinical translation of biomarker information owing to its high throughput and accuracy in the quantitative analysis of patient-derived proteomes. Using SIL internal standards ensures the utmost level of confidence in detection and precision in targeted MS experiments. For successfully establishing assays based on targeted proteomics, it is crucial to secure broad coverage when selecting the SIL standard peptide panel. However, cysteinyl peptides have often been excluded because of cysteine's high chemical reactivity. To address this limitation, a new cysteine building block was developed by incorporating a sulfhydryl group configured with an S-carbamidomethyl group, which is commonly used in proteome sampling. This compound was found to be chemically stable and applicable to a variety of solid-phase peptide synthesis (SPPS) campaigns. Furthermore, a direct comparison of the synthesized SIL peptides and tryptic endogenous peptides demonstrated the potential utility of an SPPS flow based on the new cysteine building block for improving the success of targeted proteomic applications.

Evaluation of Lipid Extraction Protocols for Untargeted Analysis of Mouse Tissue Lipidome.

Lipidomics refers to the full characterization of lipids present within a cell, tissue, organism, or biological system. One of the bottlenecks affecting reliable lipidomic analysis is the extraction of lipids from biological samples. An ideal extraction method should have a maximum lipid recovery and the ability to extract a broad range of lipid classes with acceptable reproducibility. The most common lipid extraction relies on either protein precipitation (monophasic methods) or liquid-liquid partitioning (bi- or triphasic methods). In this study, three monophasic extraction systems, isopropanol (IPA), MeOH/MTBE/CHCl3 (MMC), and EtOAc/EtOH (EE), alongside three biphasic extraction methods, Folch, butanol/MeOH/heptane/EtOAc (BUME), and MeOH/MTBE (MTBE), were evaluated for their performance in characterization of the mouse lipidome of six different tissue types, including pancreas, spleen, liver, brain, small intestine, and plasma. Sixteen lipid classes were investigated in this study using reversed-phase liquid chromatography/mass spectrometry. Results showed that all extraction methods had comparable recoveries for all tested lipid classes except lysophosphatidylcholines, lysophosphatidylethanolamines, acyl carnitines, sphingomyelines, and sphingosines. The recoveries of these classes were significantly lower with the MTBE method, which could be compensated by the addition of stable isotope-labeled internal standards prior to lipid extraction. Moreover, IPA and EE methods showed poor reproducibility in extracting lipids from most tested tissues. In general, Folch is the optimum method in terms of efficacy and reproducibility for extracting mouse pancreas, spleen, brain, and plasma. However, MMC and BUME methods are more favored when extracting mouse liver or intestine.

Stable Isotope Dilution LC/ESI-SRM/MS Analysis for Highly Polar Bioactive Dipeptides Found in Fermented Brown Rice Product using a Porous Graphitic Carbon Column

In this study, a validated analytical method based on liquid chromatography/electrospray ionization-selected reaction monitoring/mass spectrometry (LC/ESI-SRM/MS) was developed for dipeptides in a functional food, fermented brown rice and rice bran with Aspergillus oryzae (FBRA). The aim of this study was to screen and quantify dipeptides in the water-soluble fraction of FBRA. There are few studies focusing on dipeptides in FBRA, even though FBRA is expected to contain bioactive peptides: FBRA or its aqueous extract has shown variety of biological activities. An anti-hypertensive dipeptide was found in similar rice bran products digested by thermolysin. Dipeptides are the smallest class of peptides, and many show a variety of biological activities. Some bioactive peptides are widely found in fermented foods. However, because dipeptides are generally too polar to be retained on versatile LC columns, such as octadecylsilyl columns, dipeptides have been often overlooked and not quantified because of the analytical difficulties. The LC was performed using a porous graphitic carbon column with the mobile phases of 0.1% formic acid/0.1% trifluoroacetic acid/1% tetrahydrofuran in water and acetonitrile. The stable isotope-labeled internal standards and SRM for dipeptides made it possible to develop a validated method. Five dipeptides were identified and quantified in FBRA in much higher concentrations than related materials (malted brown rice, rice bran, brown rice, and white rice) as follows: Ile-Arg, 82.1 µg/g; Ala-Phe, 27.8 µg/g; Ala-Tyr, 31.6 µg/g; Val-Phe, 46.3 µg/g; and Ile-Tyr, 49.9 µg/g. This method was simple and robust and would be applicable for other functional foods.Graphical