LC-MS Method Research Articles

Bio-analytical Estimation of Metformin Co-administered with Antidiabetic and Antihyperlipidemic Herbal Preparations by LCMS

The antidiabetic and antihyperlipidemic herbal preparations were developed in liquid and oral tablet dosage form as Saptarangi Plus Kadha (SK) and Saptarangi Tablet (ST). Streptozotocin (STZ) (45mg/kg) was injected by single intraperitoneal (i.p.) injection and diabetes was induced in Wistar rats of group II to group VII. Metformin (100mg/kg, p.o.) was co-administered orally to group IV to VII with SK and ST as a reference standard drug. There is a need of bio-analytical study and standardization of herbal preparation which is required to get approval of the regulatory agencies for the commercialization. The estimation of metformin was carried in the presence of other herbal phytoconstituents by LCMS/MS. The samples of all the groups were centrifuged and vortexed. The samples were prepared in ACN: 0.1% formic acid (70:30) and LCMS method was developed and validated. The metformin was estimated in presence of active phytoconstituents of SK and ST. SK and ST contains Salacinol and folia salaciosides E1 which was detected by LCMS at m/z 333.42 and m/z 504.63 respectively. There has been an honest effort to create and validate LCMS method for the detection of metformin in presence of phytoconstituents of SK and ST in serum.

Determination of Genotoxic Impurities in Nilotinib using LC-MS Method

A validated HPLC method was developed for the determination of Nilotinib (Nob) in pharmaceutical formulation. Isocratic elution at a flow rate of 1.0ml/min was employed on Zorbax SB C18 5µm × 4.6mm, 150mm, or similar is used for this chromatography analysis and the column temperature is maintained as ambient. Mobile phase was a mixture of 2.00ml of acetic acid in 2000ml of water as mobile phase A and Acetonitrile 100% as mobile phase B was used. Rate of flow is identified at 1.3ml/min. a 50.0µl sample was injected. The run time is 20 minutes to Sample, blank, system suitability, and sensitivity solution. For the diluted standard solution, it is 60 minutes. The retention time was 1.30minutes. The% R.S.D Nob is identified. The mean percentage recovery for Nob is found within the specification limit. Due to this reason this proposed LC-MS method is successfully adopted to routine quality control analysis in formulations.

High-Throughput Acoustic Ejection Mass Spectrometry with Adjustable Signal Durations.

High-throughput mass spectrometry (MS) has witnessed rapid advancements and has found extensive applications across various disciplines. It enables the fast and accurate analysis of large sample sets, delivering a 10-fold or greater enhancement in analytical throughput when compared to conventional LC-MS methods. However, the signal duration in these high-throughput MS technologies is typically confined to a narrow range, presenting challenges for workflows demanding prolonged signal durations. In this study, we introduce a method that enables precise modulation of the signal duration on an acoustic ejection mass spectrometry (AEMS) system while ensuring high signal reproducibility. This flexibility allows for simultaneous and precise analysis of a significantly greater number of MS/MS transitions in high-throughput MS environments. Additionally, it offers a unique approach for parameter optimization and method development with minimal sample volume requirements. This advancement enhances the efficiency of MS-based analyses across diverse applications and facilitates broader utilization of MS technologies in high-throughput settings, including data-dependent acquisition (DDA) and data-independent acquisition (DIA).

Dendrobium officinale phenolic extract maintains proteostasis by regulating autophagy in a Caenorhabditis elegans model of Alzheimer's disease

Alzheimer's disease (AD) is a progressive neurodegenerative disease, and accumulating evidence suggested that proteostatic imbalance is a key feature of the disease. Traditional Chinese medicine exhibits a multi-target therapeutic effect, making it highly suitable for addressing protein homeostasis imbalance in AD. Dendrobium officinale is a traditional Chinese herbs commonly used as tonic agent in China. In this study, we investigated protection effects of D. officinale phenolic extract (SH-F) and examined its underlying mechanisms by using transgenic Caenorhabditis elegans models. We found that treatment with SH-F (50 μg/mL) alleviated Aβ and tau protein toxicity in worms, and also reduced aggregation of polyglutamine proteins to help maintain proteostasis. RNA sequencing results showed that SH-F treatment significantly affected the proteolytic process and autophagy–lysosomal pathway. Furthermore, we confirmed that SH-F showing maintainance of proteostasis was dependent on bec-1 by qRT-PCR analysis and RNAi methods. Finally, we identified active components of SH-F by LC-MS method, and found the five major compounds including koaburaside, tyramine dihydroferulate, N-p-trans-coumaroyltyramine, naringenin and isolariciresinol are the main bioactive components responsible for the anti-AD activity of SH-F. Our findings provide new insights to develop a treatment strategy for AD by targeting proteostasis, and SH-F could be an alternative drug for the treatment of AD.

A Rapid, Selective and Sensitive Electrospray Ionization Assisted LC-MS Method for Ranolazine and Identification of Its Two Potential Genotoxic Impurities

A Rapid, Selective and Sensitive Electrospray Ionization Assisted LC-MS Method for Ranolazine and Identification of Its Two Potential Genotoxic Impurities

Milk protein polymorphisms of Aosta Valley cattle breeds

Abstract Cow’s milk proteins are encoded by highly polymorphic genes characterized by several mutations which result in different allelic variants. Each allelic variant has different possible effects on cheese-making properties and on human health. β-casein A1-A3-I-B, k-casein B and β-lactoglobulin B are supposed to influence milk cheese-making properties by increasing cheese or milk yield, by varying chemical parameters, by having small casein micelle size, and by influencing rennet coagulation time (RCT) and curd-firming rate (CFR). In addition, β-casein A1-B are also considered to be a risk factor for different health diseases such as ischemic heart disease (IHD), type 1 diabetes (T1D), decreased glutathione (GSH) concentration, and milk intolerance. An LC-MS method was applied to profile, for the first time, the main milk proteins genetic variants from Aosta Valley autochtonous cattle breeds. Analyses performed on milk collected from bovines of three cattle breeds (Red Pied – VRP, Black Pied – VNP and Chestnut – CAS), either from IAR experimental farm or from herds of dairy-producers in Aosta Valley region, showed quite high frequencies of β-casein A2 and A3/I, and low frequencies of β-casein A1. Moreover, low frequencies of β-casein B in VRP breed and high frequencies of the same variant in CAS, and VNP breeds have been found. As far as k-casein is concerned allelic variant B is the most diffused in VRP while allelic variant A in VNP and CAS breeds. Finally, β-lactoglobulin most diffused allelic variant for all breeds is B. Results suggest that Aosta Valley milk has good cheese-making properties and good frequencies of β-casein A2 which may be related to beneficial effects on human health. In light of these results, it is important to develop breeding programs which take into consideration milk proteins polymorphisms to further increase the milk suitability for cheese-making process and to decrease the presence of β-casein A1 and B in drinking milk which can be a risk factor for human health.

Abstract P184: Association of Plasma Lipid Species With Lipoprotein Insulin Resistance Score Before and After Exercise Training

Background: The lipoprotein insulin resistance (LP-IR) score has been shown to assess insulin resistance, predict future type 2 diabetes, and improve with regular exercise. The lipidomic profile is known to differ with insulin resistance and type 2 diabetes, but few studies have examined lipidome associations with LP-IR, particularly in response to an exercise intervention. Methods: Plasma lipids were measured using the C8-positive LC-MS method in 671 participants from the HERITAGE Family Study (56% Female, 35% Black, 35.2 yrs) before and after a 20-week exercise intervention. LP-IR, a weighted index of six lipoprotein parameters, was measured before and after training through nuclear magnetic resonance spectroscopy (Labcorp, NC). Linear mixed models were used to test the associations of 193 known plasma lipids with LP-IR before and after exercise training. All models were adjusted for age, sex, and race, while post-training models also adjusted for baseline lipid and baseline LP-IR. Results: A total of 162 lipids (84%) were associated (FDR<0.05) with LP-IR at baseline ( Figure 1 ). The top positive associations were found for TG species, while cholesterol ester species showed the top inverse associations. Following training, the change in 143 (74%) lipids were associated (FDR<0.05) with change in LP-IR. There were 129 lipid species associated with both baseline and changes in LP-IR, while 33 were only associated with baseline measures and 14 only associated with changes in LP-IR following exercise ( Figure 1 ). Conclusions: We found that most lipids were associated with LP-IR regardless of exercise training status. Importantly, we identified a subset of lipids that were only associated with changes in LP-IR, which may represent exercise responsive biomarkers of exercise induced changes in LP-IR. However, further research is needed to identify the biological mechanisms connecting the changes in these lipid species and metabolic changes following exercise training.

Abstract P185: Plasma Lipidome Signature of Visceral Fat Reveals Heterogenous Cardiometabolic Risk Profiles

Background: Excessive visceral fat is associated with metabolic alterations and is a causal risk factor for CVD. The plasma lipidome is altered in obesity and lipidome signatures of BMI and obesity have been identified. However, few studies have examined the plasma lipidome in relation to visceral fat. Methods: Plasma lipids were measured using the C8-positive LC-MS method in 671 participants from the HERITAGE Family Study (56% Female, 35% Black, 35 yrs). Visceral fat was measured using CT scans. Linear mixed models were used to test the associations of 193 known plasma lipids with visceral fat adjusting for age, sex, race, and BMI. A FDR<5% was used to determine significance. Results: In individual models, 156 lipid species were significantly associated with visceral fat, with 109 species associated after additional adjustment for BMI ( Fig 1 ). The top positively associated lipids were triglycerides, while cholesterol esters showed the strongest inverse associations with visceral fat. A LASSO regression model retained 73 lipids and explained 78.5% of the variance in visceral fat. Delta visceral fat was calculated as the difference between predicted (from LASSO) and measured visceral fat. Examining quartiles of delta visceral fat showed that discordance between predicted and actual visceral fat was associated with differing cardiometabolic profiles independent of age, sex, race, visceral fat, and BMI. Individuals with higher delta visceral fat (Q4, overpredicted) had significantly (p<1.0x10 -04 ) higher levels of TG, apoB, total cholesterol, LDL-C, and fasting insulin and lower levels of large HDL particles and LPL activity compared to those with lower delta visceral fat (Q1, underpredicted). Conclusions: The plasma lipidome is widely associated with visceral fat levels. A lipidome-based visceral fat score may provide additional information over measured visceral fat for assessment of cardiometabolic health. Further studies are needed to test and validate the clinical utility of such lipidome-based scores.

Abstract P183: Cholesterol Ester 18:2 is an Exercise Inducible Metabolite Associated With a Favorable Cardiometabolic Profile

Background: Prospective cohort studies have shown plasma cholesterol ester (CE) 18:2 is inversely associated with all-cause and cardiovascular mortality. However, less is known about the association of CE18:2 with cardiometabolic risk factors and whether it is responsive to regular exercise. Methods: A total of 193 known plasma lipids, including 11 CEs, were measured using the C8-positive LC-MS method in 671 participants from the HERITAGE Family Study (56% Female, 35% Black, 35 yrs) before and after a 20-week exercise intervention. Linear mixed models were used to test the associations of all lipids with 6 cardiometabolic traits at baseline and post-training adjusting for age, sex, and race, with post-training models also adjusted for baseline lipid and trait values. Paired t-tests were used to test the difference in CE18:2 abundance before and after exercise training. Results: Out of the 193 lipids, CE18:2 was consistently among the top 2 associations with each cardiometabolic trait at baseline ( Table 1 ). Levels of CE18:2 were inversely associated with triglycerides, visceral fat, glycA, and small LDL, while positively associated with HDL-C and insulin sensitivity. Importantly, CE18:2 abundance was increased following the exercise intervention (p=0.0003). Exercise-induced change in CE18:2 was significantly (FDR<0.05) associated with concomitant changes in all traits except visceral fat in the same directions as baseline, with CE18:2 among the top 3 associations with each training response trait ( Table 1 ). Conclusions: Plasma CE18:2 levels were associated with cardiometabolic traits in a favorable direction both before and after an exercise intervention. Thus, this plasma lipid may be an exercise inducible metabolite that is indicative of improvements in cardiometabolic health. However, additional studies are needed to confirm the observed findings and determine the mechanisms underlying these beneficial associations.

Crystal structure, hydrogen bonding interactions, Hirshfeld surfaces, energy frameworks, and DFT calculation of Diethyl 3-(4-substitutedbenzoyl)indolizine-1,2-dicarboxylates

The diethyl 3-(4-substitutedbenzoyl)indolizine-1,2-dicarboxylates have been synthesized by reacting the electron-deficient diethyl but-2-ynedioate with 1-(2-(4-substitutedphenyl)-2-oxoethyl)pyridin-1-ium bromide, the intermediates obtained from the reaction of pyridine and 4-substituted phenacyl bromides. The synthesized compounds were investigated by FT-IR, NMR (1H and 13C), LC-MS, and elemental analysis spectroscopic methods along with PXRD. The molecular structures were determined using the single crystal X-ray diffraction technique which revealed that 5a and 5c crystallize in triclinic space group P1¯. The crystal structures of 5a and 5c preferred the C-H···O hydrogen-bonded dimeric ring motifs, resembling those observed in the earlier reported 5b. Furthermore, various computational analyzes such as Hirshfeld surface, Energy framework, and MEP analysis predicted the interaction site and inter-molecular interactions in these molecules. The FMOs analysis was carried out to obtain global reactivity parameters for the synthesized compounds (5a-c). The NCI and QTAIM studies revealed the nature as well as qualitative and quantitative description of intra and inter-molecular hydrogen bonding interactions present in these molecules and their crystal packing.

Studying the Effects of Curd as a Biocatalyst in Multicomponent Biginelli Reaction

Introduction: Dihydropyrimidinone (DHPM) is a very useful moiety with a wide range of applications. Synthesis of this moiety with environmentally friendly methods is the demand of the era. Method: In recent research, the optimisation of curd as a biocatalyst has been studied. It has been reported that it can be used directly in the multicomponent Biginelli synthesis of ethyl 6-methyl-2- oxo-4-benzyl-1,2,3,4-tetrahydropyrimidine-5-carboxylates under mild reaction conditions. Result: The chemical structures of synthesized compounds were characterized by physicochemical and analytical methods (1H NMR, IR and LC-MS methods) and it was found that curd is suitable for a vast variety of aromatic aldehydes to obtain the corresponding DHPMs. Conclusion: This new eco-friendly approach can be implemented to synthesize DHPMs for different applications.

Metabolomic Profiling Reveals the Quality Variations in Citri Reticulatae Pericarpium (Citrus reticulata Blanco cv. Chachiensis) with Different Storage Ages in Response to "Candidatus Liberibacter Asiaticus" Infection.

Citri Reticulatae Pericarpium, especially the pericarp of Citrus reticulata Blanco cv. Chachiensis (PCRC), is an important edible and medicinal ingredient for health and pharmacological properties. Citrus Huanglongbing, a devastating disease that currently threatens the citrus industry worldwide, is caused by a phloem-limited alpha-proteobacterium, "Candidatus Liberibacter asiaticus" (CLas). The industry of cultivar Chachiensis has been suffering from HLB. Although HLB affected the quality of citrus fruit, whether the quality of PCRC was affected by HLB remains unclear. In this study, we compared the metabolite profiles between HLB-affected and healthy PCRC from three sources: fresh, 6-month-old, and 9-year-old PCRC, through the untargeted LC-MS method. Compared to healthy controls, various types of bioactive compounds, mainly flavonoids, terpenoids, alkaloids, coumarins, polysaccharides, and phenolic acids, accumulated in HLB-affected PCRC, especially in the HLB-affected 9-year PCRC. In particular, isorhamnetin, isoliquiritigenin, luteolin 7-O-beta-D-glucoside, limonin, geniposide, pyrimidodiazepine, scoparone, chitobiose, m-coumaric acid, malonate, and pantothenic acid, which contributed to the pharmacological activity and health care effects of PCRC, were highly accumulated in HLB-affected 9-year-old PCRC compared to the healthy control. Multibioassay analyses revealed that HLB-affected 9-year-old PCRC had a higher content of total flavonoids and total polyphenols and exhibited similar antioxidant capacity as compared to healthy controls. The results of this study provided detailed information on the quality of HLB-affected PCRC.

Analyses of plasma metabolites using a high performance four-channel CIL LC-MS method and identification of metabolites associated with enteric methane emissions in beef cattle.

Reducing enteric methane (one greenhouse gas) emissions from beef cattle not only can be beneficial in reducing global warming, but also improve efficiency of nutrient utilization in the production system. However, direct measurement of enteric methane emissions on individual cattle is difficult and expensive. The objective of this study was to detect plasma metabolites that are associated with enteric methane emissions in beef cattle. Average enteric methane emissions (CH4) per day (AVG_DAILYCH4) for each individual cattle were measured using the GreenFeed emission monitoring (GEM) unit system, and beef cattle with divergent AVG_DAILYCH4 from Angus (n = 10 for the low CH4 group and 9 for the high CH4 group), Charolais (n = 10 for low and 10 for = high), and Kinsella Composite (n = 10 for low and 10 for high) populations were used for plasma metabolite quantification and metabolite-CH4 association analyses. Blood samples of these cattle were collected near the end of the GEM system tests and a high performance four-channel chemical isotope labeling (CIL) liquid chromatography (LC) mass spectrometer (MS) method was applied to identify and quantify concentrations of metabolites. The four-channel CIL LC-MS method detected 4235 metabolites, of which 1105 were found to be significantly associated with AVG_DAILYCH4 by a t-test, while 1305 were significantly associated with AVG_DAILYCH4 by a regression analysis at p<0.05. Both the results of the t-test and regression analysis revealed that metabolites that were associated with enteric methane emissions in beef cattle were largely breed-specific whereas 4.29% to 6.39% CH4 associated metabolites were common across the three breed populations and 11.07% to 19.08% were common between two breed populations. Pathway analyses of the CH4 associated metabolites identified top enriched molecular processes for each breed population, including arginine and proline metabolism, arginine biosynthesis, butanoate metabolism, and glutathione metabolism for Angus; beta-alanine metabolism, pyruvate metabolism, glycolysis / gluconeogenesis, and citrate cycle (TCA cycle) for Charolais; phenylalanine, tyrosine and tryptophan biosynthesis, phenylalanine metabolism, arginine biosynthesis, and arginine and proline metabolism for Kinsella Composite. The detected CH4 associated metabolites and enriched molecular processes will help understand biological mechanisms of enteric methane emissions in beef cattle. The detected CH4 associated plasma metabolites will also provide valuable resources to further characterize the metabolites and verify their utility as biomarkers for selection of cattle with reduced methane emissions.

Metabolomics characterization of two saffron from Iran and China using GC–MS and LC–MS methods

Saffron is a medicinally important species of the Iridaceae family. Saffron’s quality depends on the concentration of secondary metabolites. In this study, GC–MS and LC–MS were utilized to dissect the different metabolites profile of two saffron produced in Iran and China. As a result, a total of 325 differential metabolites (237 from LC–MS, 88 from GC–MS) were identified. These differential metabolites were grouped into the following categories such as lipids, alkaloids, amino acids, terpenoids, organic acids, and flavonoids in saffron from the LC–MS analysis. The differential metabolites mainly concentrated on metabolic pathways, nucleotide metabolism, purine metabolism, and flavonoid biosynthesis. In Iran vs. China, the category of flavonoids includes Styrene, Apigenin-7-O-(6''-p-Coumaryl)glucoside, Houpulin F, Apigenin-5-O-glucoside, Tiglic acid; 2-Methyl-2-Butenoic Acid, p-Cymene, and 2,4-Nonadienal, (E,E)-. The terpenoids including downregulated Cafestol, rhodomollein XI, inumakiol G, D-Limonene, Bicyclo[3.1.0]hexane, 4-methylene-1-(1-methylethyl)- and upregulated rhodomollein XI were significantly different identified in Iran vs. China. In conclusion, the saffron from different cultivation areas possess many metabolites that are beneficial for health, including lipids, alkaloids, amino acids, terpenoids, organic acids, and flavonoids. Thus, compared with Iran, saffron from China have a higher medicinal value. This study provides a direction for the subsequent development and utilization of saffron.

Development and Validation of Simple and Stable LCMS Method for the Quantification of Potential Genotoxic Impurities in Ozenoxacin Pure Drug and its Commercial Preparations

Ozenoxacin is an antibiotic drug prescribed to treat various skin infections caused by various bacteria. Various chemical mechanisms such as stille coupling, Buchwald–Hartwig coupling, cyclization and saponification are involved during the process of synthesis of Ozenoxacin. In the process of synthesis, there is a possibility of the formation of related impurities and among them, some are genotoxic impurities. To date, in literature, there is no method reported for analysing Potential Genotoxic Impurities (PGIs) in Ozenoxacin and hence this study was initiated to develop an LCMS method for quantification of two genotoxic impurities of Ozenoxacin viz., nitroso impurity, ester impurity. The analytes were resolved on Alltima C18 column (150×4.6mm; 5 μm particle size) using 0.01 mM ammonium acetate at pH 4.8 and methanol in 80:20 (v/v) at 0.5 mL/min flow rate and 10 μ sample injection volume. The multiple reaction monitoring of the mass fragments confirms the parent ion at m/z of 364, 393 and 496 for Ozenoxacin, Nitroso and Ester impurity respectively with characteristic product ion at m/z 196. The method has a linearity range of 0.05 μg/mL to 1.0 μg/mL for three analytes with detection limits of 0.015, 0.011 and 0.015 μg/mL for Ester impurity, Ozenoxacin and Nitroso impurity respectively. The method was validated and produces acceptable results and can successfully separate the potential genotoxic impurities in spiked commercial samples. Based on the findings, it was concluded that this method can be practically useful for the identification and quantification of potential genotoxic impurities and may apply to the safe use of Ozenoxacin in clinical treatment.

Can Small Molecules Provide Clues on Disease Progression in Cerebrospinal Fluid from Mild Cognitive Impairment and Alzheimer's Disease Patients?

Alzheimer's disease (AD) is a complex and multifactorial neurodegenerative disease, which is currently diagnosed via clinical symptoms and nonspecific biomarkers (such as Aβ1-42, t-Tau, and p-Tau) measured in cerebrospinal fluid (CSF), which alone do not provide sufficient insights into disease progression. In this pilot study, these biomarkers were complemented with small-molecule analysis using non-target high-resolution mass spectrometry coupled with liquid chromatography (LC) on the CSF of three groups: AD, mild cognitive impairment (MCI) due to AD, and a non-demented (ND) control group. An open-source cheminformatics pipeline based on MS-DIAL and patRoon was enhanced using CSF- and AD-specific suspect lists to assist in data interpretation. Chemical Similarity Enrichment Analysis revealed a significant increase of hydroxybutyrates in AD, including 3-hydroxybutanoic acid, which was found at higher levels in AD compared to MCI and ND. Furthermore, a highly sensitive target LC-MS method was used to quantify 35 bile acids (BAs) in the CSF, revealing several statistically significant differences including higher dehydrolithocholic acid levels and decreased conjugated BA levels in AD. This work provides several promising small-molecule hypotheses that could be used to help track the progression of AD in CSF samples.

Rapid analysis of trace pollutants chlorates, perchlorates, and four xanthates in water based on IC-MS combined technology

Water, as the source of life, plays a crucial role in life activities. It also acts as a solvent, allowing toxic and harmful substances to migrate through the water environment. In this study, an analytical method based on ion chromatography-mass spectrometry (IC-MS) is established for the direct determination of trace chlorates, perchlorates, and four xanthates in water. The Dionex IonPac AS20 chromatography column is used as the analytical column, and the CR-TC device automatically generates KOH eluent and sets up a multi-step gradient program for elution. The Dionex ADRS 600 self-circulating regeneration ion suppressor (2 mm) generates H+ through electrolysis of water, replacing metal ions in the sample solution and eluent, enabling direct tandem ion chromatography and mass spectrometry. The water sample is directly measured by IC-MS after being filtered by a hydrophilic polytetrafluoroethylene (PTFE) filter head. The results indicate that chlorate and perchlorate show good linearity in the range of 0.1–100 ng mL−1, with a quantification limit of 0.3 μg L−1. The four xanthates exhibit a good linear relationship in the range of 0.5–500 ng mL−1, and the quantification limit can be reduced to 1 μg L−1. The method offers advantages such as high recovery rate, good stability, and strong reproducibility. It shows high consistency with the results detected by the LC-MS method, but with the added benefits of being more advanced and time-saving. This innovative technology provides a new detection method for trace pollutants in water, which can aid in the prevention and control of pollutants in the water environment.

Endogenous monoclonal immunoglobulins analyzed using the EXENT® solution and LC-MS

IntroductionThe EXENT® Solution, a fully automated system, is a recent advancement for identifying and quantifying monoclonal immunoglobulins in serum. It combines immunoprecipitation with MALDI-TOF mass spectrometry. Compared to gel-based methods, like SPEP and IFE, it has demonstrated the ability to detect monoclonal immunoglobulins in serum at lower levels. In this study, samples that tested negative using EXENT® were reflexed to LC-MS to determine if the more sensitive LC-MS method could identify monoclonal immunoglobulins missed by EXENT®. ObjectivesTo assess whether monoclonal immunoglobulins that are not detected by EXENT® can be detected by LC-MS using a low flow LC system coupled to a Q-TOF mass spectrometer. MethodsSamples obtained from patients confirmed to have multiple myeloma (MM) were diluted with pooled polyclonal human serum and analyzed using EXENT®. If a specific monoclonal immunoglobulin was not detected by EXENT®, the sample was then subjected to analysis by LC-MS. For the LC-MS analysis, the sample eluate, obtained after the MALDI-TOF MS spotting step, was collected and transferred to an autosampler tray for subsequent analysis using LC-MS. ConclusionLC-MS has the capability to detect monoclonal immunoglobulins that are no longer detected by EXENT®. Reflexing samples to LC-MS for analysis does not involve additional sample handling, allowing for a faster time-to-result compared to current approaches, such as Next-Generation Sequencing, Next-Generation Flow, and clonotypic peptide methods. Notably, LC-MS offers equivalent sensitivity in detecting these specific monoclonal immunoglobulins.

MetabCombiner 2.0: Disparate Multi-Dataset Feature Alignment for LC-MS Metabolomics.

Liquid chromatography-high-resolution mass spectrometry (LC-HRMS), as applied to untargeted metabolomics, enables the simultaneous detection of thousands of small molecules, generating complex datasets. Alignment is a crucial step in data processing pipelines, whereby LC-MS features derived from common ions are assembled into a unified matrix amenable to further analysis. Variability in the analytical factors that influence liquid chromatography separations complicates data alignment. This is prominent when aligning data acquired in different laboratories, generated using non-identical instruments, or between batches from large-scale studies. Previously, we developed metabCombiner for aligning disparately acquired LC-MS metabolomics datasets. Here, we report significant upgrades to metabCombiner that enable the stepwise alignment of multiple untargeted LC-MS metabolomics datasets, facilitating inter-laboratory reproducibility studies. To accomplish this, a "primary" feature list is used as a template for matching compounds in "target" feature lists. We demonstrate this workflow by aligning four lipidomics datasets from core laboratories generated using each institution's in-house LC-MS instrumentation and methods. We also introduce batchCombine, an application of the metabCombiner framework for aligning experiments composed of multiple batches. metabCombiner is available as an R package on Github and Bioconductor, along with a new online version implemented as an R Shiny App.

Immunocapture LC-MS methods for pharmacokinetics of large molecule drugs.

Implementation of immunocapture LC-MS methods to characterize the pharmacokinetic profile of large molecule drugs has become a widely used technique over the past decade. As the pharmaceutical industry strives for speediness into clinical development without jeopardizing quality, robust assays with generic application across the pipeline are becoming instrumental in bioanalysis, especially in early-stage development. This review highlights the capabilities and challenges involved in hybrid immunocapture LC-MS techniques and its continued applications in nonclinical and clinical pharmacokinetic assay design. This includes a comparison of LC-MS-based approaches to conventional ligand-binding assays and the driving demands in large molecule drug portfolios including growing sensitivity requirements and the unique challenges of new modalities requiring innovation in the bioanalytical laboratory.