HILIC Column Research Articles

Quantitation of Copper Tripeptide in Cosmetics via Fabric Phase Sorptive Extraction Combined with Zwitterionic Hydrophilic Interaction Liquid Chromatography and UV/Vis Detection

The increasing demand for effective cosmetics has driven the development of innovative analytical techniques to ensure product quality. This work presents the development and validation of a zwitterionic hydrophilic interaction liquid chromatography method, coupled with ultraviolet detection, for the quantification of copper tripeptide in cosmetics. A novel protocol for sample preparation was developed using fabric phase sorptive extraction to extract the targeted analyte from the complex cosmetic cream matrix, followed by chromatographic separation on a ZIC®-pHILIC analytical column. A thorough investigation of the chromatographic behavior of the copper tripeptide on the HILIC column was performed during method development. The mobile phase consisted of 133 mM ammonium formate (pH 9, adjusted with ammonium hydroxide) and acetonitrile at a 40:60 (v/v) ratio, with a flow rate of 0.2 mL/min. A design of experiments (DOE) approach allowed precise adjustments to various factors influencing the extraction process, leading to the optimization of the fabric phase sorptive extraction protocol for copper tripeptide analysis. The method demonstrated excellent linearity over a concentration range of 0.002 to 0.005% w/w for copper tripeptide, with a correlation coefficient exceeding 0.998. The limits of detection and quantitation were 5.3 × 10−4% w/w and 2.0 × 10−3% w/w, respectively. The selectivity of the method was verified through successful separation of copper tripeptide from other cream components within 10 min, establishing its suitability for high-throughput quality control of cosmetic formulations.

Development of a rapid and robust hydrop interaction liquid chromatography tandem mass spectrometry method for the detection of 13 endogenous amino acids as well as trimethylamine oxide in serum and tissues of the mice.

This work aimed to establish an HILIC-MS/MS method to simultaneously determine the levels of 13 endogenous amino acids and trimethylamine oxide in the biological samples from the mice. Electrospray ion source was used for the analysis of mass spectrometry. The 20 min separation was applied in a Dikma Inspire Hilic column (2.1 × 100.0mm, 3μM). Positive ion mode under an MRM model gave a satisfying response value. The limits of quantitation were evaluated by accuracy from -12.59% to 7.89% and precision from 1.77% to 14.00% as well as acceptable interday and intraday precision, matrix effect, recovery, and stability. Later, the assay was successfully used to measure the concentrations of the determinands in the biological samples. Individual and tissue distribution differences for these metabolites were observable. The amino acids had a consistent highest content in the spleens, while the lowest levels were found in the livers. Alanine was the most abundant amino acid in the serum, and taurine kept the highest content in all of the tissues. Trimethylamine oxide remained low level, especially in the liver samples.

Per aqueous liquid chromatography of Radix hedysari polysaccharides and Au nanoparticles co-functionalized stationary phase and its application in the determination of iridoids and phenylethanols

BackgroundHydrophilic Interaction Liquid Chromatography (HILIC) is an outstanding strategy for the challenging analysis of hydrophilic and polar components. Nevertheless, analysis under HILIC mode typically consumes 70%–95 % acetonitrile with the disadvantage of high analytical costs, being environmentally unfriendly and causing biohazards, which is not in line with the concept of green chromatography. Research has shown that Per Aqueous Liquid Chromatography (PALC) simultaneously emphasizes efficient analytical performance for hydrophilic analytes and green analytical concepts. The development of new PALC stationary phases with superior performance is necessary. ResultsIn this paper, silanized silica was sequentially subjected to esterification reaction, polymerization reaction and covalent bonding through five steps to obtain SiO2-RHP-AuNPs material, which was prepared as a novel stationary phase for PALC. Comprehensive characterization of the materials by means of Fourier transform infrared spectroscopy, Transmission scanning electron microscope, Elemental analysis and Thermogravimetric analysis showed the successful bonding of the functionalized groups on the original silica. The polymeric stationary phase based on Radix hedysari polysaccharide and Au nanoparticles had higher density of hydroxyl and ester functionalized groups. The Au nanoparticles upgraded their mesoporous structure and thermal stability, providing exceptional chromatographic performance and selectivity for chromatographic analysis. The influence of mobile phase water content, salt concentration, pH and column temperature on the retention behavior was evaluated. The novel Column was found to exhibit a dual mechanism of hydrophobic interactions/ion exchange interactions in a mobile phase with high water content. Significance and noveltyThe separation efficiency and selectivity of SiO2-RHP-AuNPs columns for synthetic pigments and organic acids in PALC mode were superior to those of commercial HILIC and C18 columns. In addition, a method for the determination of seven active ingredients in Fructus Ligustri Lucidi by SiO2-RHP-AuNPs column in PALC mode was developed. The method had good stability, reproducibility and accuracy, which was capable of realizing the quality evaluation of Chinese Materia Medicas.

Determination of dimethyl sulfate genotoxic impurity in pantoprazole sodium sesquihydrate by derivatization method and UPLC/MS

In this study, a new method has been developed and, the method has been validated to analyze dimethyl sulfate (DMS) which known as a genotoxic impurity. DMS has been detected in trace levels within the drug substance pantoprazole sodium sesquihydrate using the derivatization method. Triethylamine has been used as derivatization reagent. Due to the highly polar nature of the derivatization product, which is a quaternary ammonium ion, a polar retention Hilic column has been used for chromatographic separation. The Waters QDA detector has been used in the single ion recording (SIR) mode at m/z 116 for the detection of the quaternary ammonium ion. In this study, the recovery rates have been achieved within the range of 96.46–105.98%. The LOD has been determined as 1.94E-07 mg/mL, and the LOQ has been determined as 6.46E-07 mg/mL. From the results, it can be said that the improved method in this study, would enables fast and reliable solutions for routine quality control analyses for DMS in API companies that especially is producing PSS.

Simultaneous determination of canonical purine metabolism using a newly developed HILIC-MS/MS in cultured cells

Purine metabolism acts as the core role in human metabolic network. It offers purine metabolites as raw material for building blocks in cell survival and proliferation. Purine metabolites are the most abundant metabolic substrates in organisms. There are few reports to simultaneously quantify canonical purine metabolism in cells. A novel hydrophilic interaction liquid chromatography coupled with mass spectrometry (HILIC-MS/MS) method was developed to simultaneously determine purines profile in biological samples. Chromatographic separation was achieved using a HILIC (Waters Xbridge™ Amide) column. Different optimizing chromatographic conditions and mass spectrometric parameters were tested in order to provide the best separation and the lowest limit of quantification (LLOQ) values for targeted metabolites. The validation was evaluated according to the Food and Drug Administration guidelines. The limit of determination (LOD) and the LOQ values were in the range of 0.02–8.33 ng mL−1 and 0.1–24.5 ng mL−1, respectively. All calibration curves displayed good linear relationship of with excellent correlation coefficient (r) ranging from 0.9943 to 0.9999. Both intra-day and inter-day variability were below 15 %, respectively. Trueness, expressed as relative error, was always within ±15 %. In addition, no derivatization procedure and ion-pair reagents are in need. The innovated approach demonstrates high sensitivity, strong specificity, and good repeatability, making it suitable for absolute quantitative studies of canonical purine metabolism in cultured cells.

Quantification of relevant metabolites in apoptotic bodies from HK-2 cells by targeted metabolomics based on liquid chromatography-tandem mass spectrometry

BackgroundApoptotic bodies play an important role in the cellular communication as a consequence of the great variety of biomolecules they harbor. There is evidence that 1st generation apoptotic bodies from HK-2 cells induced by cisplatin or UV light trigger apoptosis in naïve HK-2 cells whereas 2nd generation apoptotic bodies activate cell proliferation showing an opposite effect. Thus, the development of new analytical strategies to quantify the changes in the involved metabolites is imperative to shed light on the biological mechanisms which trigger apoptosis and cell proliferation. ResultsA LC-(Q-Orbitrap)MS method has been developed to quantify the metabolites unequivocally identified in the apoptotic body fluid from HK-2 cells in our previous works based on untargeted metabolomics. Thus, two different columns and gradients were tested and the HILIC column was selected taking into account the retention times and chromatographic separation. Also, different normal collision energies were tested for each metabolite and the parallel reaction monitoring was chosen to carry out the quantitative analysis. Once the method was optimized, it was evaluated in terms of linearity, limits of detection and quantification, matrix effects, accuracy, and precision, for each metabolite. Limits of detection ranged from 0.02 to 1.4 ng mL−1. A total of 9 relevant metabolites proposed as potential biomarkers to reveal metabolic differences among apoptotic bodies from HK-2 cells were quantified and some insights about the biological relevance were discussed. SignificanceThe first targeted metabolomics methodology enabling the quantification of relevant metabolites in apoptotic bodies from HK-2 cells was developed using LC-(Q-Orbitrap)MS. Pyridoxine, kynurenine, and creatine concentrations were determined in apoptotic bodies from HK-2 cells treated with cisplatin and UV light. Phenylacetylglycine, hippuric acid, butyrylcarnitine, acetylcarnitine, carnitine, and phenylalanine were determined in 1st and 2nd generation apoptotic bodies from HK-2 cells treated with cisplatin. Concentrations determined were useful to establish their biological role in the metabolism.

LC-ESI-MS/MS methods for the quantification of hydrazine mono lactose adduct and hydrazine di lactose adduct in isosorbide dinitrate and hydralazine hydrochloride tablets

Isosorbide dinitrate and hydralazine hydrochloride tablets treat heart failure in addition to conventional therapy, prolong hospitalization for heart failure, and enhance patient-reported functional status. LC-MS/MS methods developed and validated separately for the quantification of hydrazine mono-lactose adduct and hydrazine di-lactose adduct impurities in the isosorbide and hydralazine hydrochloride tablets. Separation of hydrazine mono-lactose adduct impurity achieved on ZIC-HILIC (100 × 4.6 mm, 5 µm) column with 0.1% formic acid in water as mobile phase A and acetonitrile as mobile phase B. The HPLC method gradient elution is; Tmin/% of B: 0/90, 15/40, 15.1/90, and 20/90. The flow rate is 1.0 mL per minute, and the injection volume is 20 µL. Separation of hydrazine di-lactose adduct impurity achieved on Inertsil HILIC (150 × 4.6 mm, 5 µm) column with 10 Mm ammonium acetate in water as mobile phase A and acetonitrile as mobile phase B. The HPLC method gradient elution is; Tmin/% of B: 0/90, 15/40, 15.1/90, and 18/90. The flow rate is 1.0 mL per minute, and the injection volume is 40 µL. Method validation has demonstrated both methods are specific, sensitive, linear, precise, accurate, stable and robust.

Pharmacokinetic assessment of low dose decitabine in combination therapies: Development and validation of a sensitive UHPLC-MS/MS method for murine plasma analysis

Decitabine is a DNA methyltransferase inhibitor used in the treatment of acute myeloid leukemia and myelodysplastic syndrome. The notion that ongoing trials are presently exploring the combined use of decitabine, with or without the cytidine deaminase inhibitor cedazuridine, and other antileukemic drugs necessitates a comprehensive understanding of pharmacokinetic properties and an evaluation of drug-drug interaction liabilities. We report here the development and validation of a sensitive UHPLC-MS/MS method for quantifying decitabine in mouse plasma, which should be useful for such studies. The method involved a one-step protein precipitation extraction, and chromatographic separation on an XBridge HILIC column using gradient elution. The method was found to be robust, accurate, precise, and sufficiently sensitive (lower limit of quantitation, 0.4 ng/mL) to determine decitabine concentrations in microvolumes of plasma from mice receiving the agent orally or intravenously in the presence or absence of cedazuridine.

Standards-Free Absolute Quantitation of Oxidizable Glycopeptides by Coulometric Mass Spectrometry.

Currently, glycopeptide quantitation is mainly based on relative quantitation due to absolute quantitation requiring isotope-labeled or standard glycopeptides which may not be commercially available or are very costly and time consuming to synthesize. To address this grand challenge, coulometric mass spectrometry (CMS), based on the combination of electrochemistry (EC) and mass spectrometry (MS), was utilized to quantify electrochemically active glycopeptides without the need of using standard materials. In this study, we studied tyrosine-containing glycopeptides, NYIVGQPSS(β-GlcNAc)TGNL-OH and NYSVPSS(β-GlcNAc)TGNL-OH, and successfully quantified them directly with CMS with a discrepancy of less than 5% between the CMS measured amount and the theoretical amount. Taking one step further, we applied this approach to quantify glycopeptides generated from the digestion of NIST mAb, a monoclonal antibody reference material. Through HILIC column separation, five N297 glycopeptides resulting from NIST mAb tryptic digestion were successfully separated and quantified by CMS for an absolute amount without the use of any standard materials. This study indicates the potential utility of CMS for quantitative proteomics research.

Metabolite profiling of chemically modified oligonucleotides in rat liver homogenates using hydrophilic interaction liquid chromatography-high resolution mass spectrometry

Chemically modified oligonucleotide is a rapidly emerging field of biotherapeutics. The evaluation of the metabolic stability of therapeutic oligonucleotides is critical. The current methods are limited to the ion-pairing reversed phase chromatographic techniques. Due to complex chemical modifications of therapeutic oligonucleotides and diverse structural changes in their metabolites, comprehensive metabolite profiling is still challenging. Therefore, an alternative analytical method for the metabolism study of oligonucleotides is urgently needed. In this study, a novel hydrophilic interaction liquid chromatography-high resolution mass spectrometry (HILIC-HRMS) method was developed and applied to the metabolism study of two chemically modified oligonucleotides, i.e., N11-GalNAc (phosphorothioate, 2′-F, 2′-OMe, and 3′-GalNAc) and N9 (phosphorothioate, 2′-F), in rat liver homogenates. Highly broad retention of metabolites on the HILIC column was achieved, with truncated metabolite having a single nucleotide being detected. A stepped normalized collisional energy (NCE) in HRMS led to characteristic c/y, d/y, and b/y fragment ion series, and diagnostic fragment ions for linker and GalNAc, which covered the whole sequence of oligonucleotides. A total number of twenty-seven and twenty-nine metabolites were identified in rat liver homogenates for N11-GalNAc and N9, respectively, by using the isotopic envelope of the precursor ion and stepped NCE-based sequence mapping data. The time-dependent production of metabolites was observed. The UCU sequence with the uniform 2′-F modification on ribose was identified as the major metabolic site for N11-GalNAc, and the phosphodiester bonds between nucleosides involving unmodified uracil were identified as the metabolic soft sites for N9. The developed HILIC-HRMS method provided a high coverage of various oligonucleotide metabolites. Characteristic fragment ions enable sequence mapping and therefore unambiguous metabolite identifications. This approach can be broadly applicable to the metabolism study of chemically modified oligonucleotides and the identification of their metabolic soft sites.

A rapid and highly sensitive HILIC-HPLC based analytical assay method for quantification of azacitidine and venetoclax in a single run

Venetoclax and Azacitidine combination has recently been approved by the USFDA to treat old people with acute myeloid leukemia. Pharmaceutical formulations of the combination of these two drugs are expected to be launched soon. Till date, an analytical method of any type has not been reported in literature for quantification of Venetoclax and Azacitidine in a single run. The current work set out to establish an HILIC-HPLC based analytical assay method for simultaneous quantification of Azacitidine and Venetoclax from the mixed sample. The method has been developed employing Poroshell HILIC column. Ammonium formate (10 mM, pH 5.0) and acetonitrile were used as a mobile phase in gradient programme. A variety of parameters, including linearity, accuracy, precision, specificity, robustness, and ruggedness were evaluated to validate the method. Azacitidine and Venetoclax were eluted at 8.8 min and 4.0 min, respectively. Method was linear over the range of 1-100 µg/mL. Recovery was found to be 98.29-99.28% and 101.84-101.95% for Azacitidine and Venetoclax, respectively. The method was reproducible with a relative standard deviation of less than two in precision studies. The developed method can suitably be employed for quantifying Venetoclax and Azacitidine in a single run from their mixed samples.

Olaris Global Panel (OGP): A Highly Accurate and Reproducible Triple Quadrupole Mass Spectrometry-Based Metabolomics Method for Clinical Biomarker Discovery.

Mass spectrometry (MS)-based clinical metabolomics is very promising for the discovery of new biomarkers and diagnostics. However, poor data accuracy and reproducibility limit its true potential, especially when performing data analysis across multiple sample sets. While high-resolution mass spectrometry has gained considerable popularity for discovery metabolomics, triple quadrupole (QqQ) instruments offer several benefits for the measurement of known metabolites in clinical samples. These benefits include high sensitivity and a wide dynamic range. Here, we present the Olaris Global Panel (OGP), a HILIC LC-QqQ MS method for the comprehensive analysis of ~250 metabolites from all major metabolic pathways in clinical samples. For the development of this method, multiple HILIC columns and mobile phase conditions were compared, the robustness of the leading LC method assessed, and MS acquisition settings optimized for optimal data quality. Next, the effect of U-13C metabolite yeast extract spike-ins was assessed based on data accuracy and precision. The use of these U-13C-metabolites as internal standards improved the goodness of fit to a linear calibration curve from r2 < 0.75 for raw data to >0.90 for most metabolites across the entire clinical concentration range of urine samples. Median within-batch CVs for all metabolite ratios to internal standards were consistently lower than 7% and less than 10% across batches that were acquired over a six-month period. Finally, the robustness of the OGP method, and its ability to identify biomarkers, was confirmed using a large sample set.

Synthesis of weak cation exchange/C18 bifunctional magnetic polymers for pretreatment and determination of glufosinate and its two metabolites in plasma samples

This study focuses on the purification and detection of glufosinate (GLUF) and its metabolites N-acetyl GLUF and MPP in plasma samples. A Dikma Polyamino HILIC column was used for the effective retention and separation of GLUF and its metabolites, and the innovative addition of a low concentration of ammonium fluoride solution to the mobile phase effectively improved the detection sensitivity of the target analytes. Monodisperse core-shell weak cation exchange (WCX)/C18 bifunctional magnetic polymer composites (Fe3O4@WCX/C18) were prepared in a controllable manner, and their morphology and composition were fully characterized. The Fe3O4@WCX/C18 microspheres were used as a magnetic solid-phase extraction (MSPE) adsorbent for the sample purification and detection of GLUF and its metabolites in plasma samples combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS). The purification conditions of Fe3O4@WCX/C18 microspheres for GLUF and its metabolites in spiked plasma samples were optimized to achieve the best MSPE efficiency. The purification mechanisms of the target analytes in plasma samples include electrostatic attraction and hydrophobic interactions. Furthermore, the effect of the molar ratio of the two functional monomers 4-VBA and 1-octadecene in the adsorbent was optimized and it shows that the bifunctional components WCX/C18 have a synergistic effect on the determination of GLUF and its metabolites in plasma samples. In addition, the present study compared the purification performance of the Fe3O4@WCX/C18 microsphere-based MSPE method with that of the commercial Oasis WCX SPE method, and the results showed that the Fe3O4@WCX/C18 microsphere-based MSPE method established in this work had a stronger ability to remove matrix interferences. Under optimal purification conditions, the recoveries of GLUF and its metabolites in plasma were 87.6–111 % with relative standard deviations (RSDs) ranging from 0.2 % to 4.8 %. The limits of detection (LODs, S/N≥3) and limits of quantification (LOQs, S/N≥10) were 0.10–0.18 μg/L and 0.30–0.54 μg/L, respectively. The MSPE-LC-MS/MS method developed in this study is fast, simple, accurate and sensitive and can be used to confirm GLUF intoxication based not only on the detection of the GLUF prototype but also on the detection of its two metabolites.

Bioanalysis of Stress Biomarkers through Sensitive HILIC-MS/MS Method: A Stride toward Accurate Quantification of MDA, ACR, and CTA.

Quantifying reactive aldehyde biomarkers, such as malondialdehyde, acrolein, and crotonaldehyde, is the most preferred approach to determine oxidative stress. However, reported analytical methods lack specificity for accurately quantifying these aldehydes as certain methodologies may produce false positive results due to harsh experimental conditions. Thus, in this research work, a novel HILIC-MS/MS method with endogenous histidine derivatization is developed, which proves to have higher specificity and reproducibility in quantifying these aldehydes from the biological matrix. To overcome the reactivity of aldehyde, endogenous histidine is used for its derivatization. The generated adduct is orthogonally characterized by NMR and LC-HRMS. The method employed a hydrophilic HILIC column and multiple reaction monitoring (MRM) to accurately quantify these reactive aldehydes. The developed method is an unequivocal solution for quantifying stress in in vivo and in vitro studies.

Highly sensitive and accurate measurement of underivatized phosphoenolpyruvate in plasma and serum via EDTA-facilitated hydrophilic interaction liquid chromatography–tandem mass spectrometry

Phosphoenolpyruvate (PEP) is an essential intermediate metabolite that is involved in various vital biochemical reactions. However, achieving the direct and accurate quantification of PEP in plasma or serum poses a significant challenge owing to its strong polarity and metal affinity. In this study, a sensitive method for the direct determination of PEP in plasma and serum based on ethylenediaminetetraacetic acid (EDTA)-facilitated hydrophilic interaction liquid chromatography–tandem mass spectrometry was developed. Superior chromatographic retention and peak shapes were achieved using a zwitterionic stationary-phase HILIC column with a metal-inert inner surface. Efficient dechelation of PEP–metal complexes in serum/plasma samples was achieved through the introduction of EDTA, resulting in a significant enhancement of the PEP signal. A PEP isotopically labelled standard was employed as a surrogate analyte for the determination of endogenous PEP, and validation assessments proved the sensitivity, selectivity, and reproducibility of this method. The method was applied to the comparative quantification of PEP in plasma and serum samples from mice and rats, as well as in HepG2 cells, HEK293T cells, and erythrocytes; the results confirmed its applicability in PEP-related biomedical research. The developed method can quantify PEP in diverse biological matrices, providing a feasible opportunity to investigate the role of PEP in relevant biomedical research.

Abstract 3495: Multi-omic landscape of squamous cell lung cancer

Abstract Patients with lung squamous cell carcinoma (LSCC) require new drug targets and improved biomarkers due to a lack of targetable genomic drivers and low response rates to immune checkpoint blockade. In a previous study, we analyzed a cohort of 108 LSCC patients by integrating DNA copy number variation, somatic mutations, RNA-sequencing, and expression proteomics. The principal discovery was the identification of three proteomic subtypes, with the majority (87%) of tumors comprising two of these subtypes. The "Inflamed" subtype showed enrichment for B-cell-rich tertiary lymphoid structures, while the "Redox" subtype exhibited enrichment for redox pathways and NFE2L2/KEAP1 alterations but had notably lower immune infiltration. We hypothesized that these subtypes would result in distinct metabolic signatures. Using ultra-high-performance liquid chromatographic separation on a HILIC column, followed by analysis on a Q Exactive HF high resolution mass spectrometer, we performed untargeted metabolomics on 87 tumors from the same LSCC proteogenomics cohort. A total of 7,392 features were obtained from this analysis, leading to the identification of 446 metabolites through m/z and retention time matching against an internal reference library. To understand if metabolomics could recapitulate our proteomic subtypes, we applied consensus clustering and non-negative matrix factorization (NMF) and assessed the resulting clusters using a Random Forest (RF) supervised classifier. Area Under the Curve (AUC) values for consensus clustering (5 clusters, AUC = 0.72), NMF (4 clusters, AUC = 0.73), and proteomics subtypes (Stewart et al. Nature communications. 2019:10:3578) (3 clusters, AUC = 0.74) suggest that metabolite abundances do indeed recapitulate the proteomic subtypes. Differential expression between Redox and Inflamed yielded 29 differentially expressed metabolites (p-value &amp;lt; 0.05 and 1.5 fold change). Glutathione, a key redox metabolite, was modestly elevated in the Redox proteomic subtype (0.58 log2 ratio, p = 1.14E-05). Notably, we identified glutathione metabolism (p = 1.29-5) and arginine biosynthesis (p = 5.22-4) as among the most significant pathways among differentially expressed metabolites. Glutathione, a key redox metabolite, was modestly elevated in the Redox proteomic subtype (0.58 log2 ratio, p = 1.14E-05). In conclusion, metabolomics recapitulates the proteomic subtypes, and there are distinct differences between these subtypes at the metabolite level. Ongoing work is developing a novel, network-based analysis framework to integrate these data quantitatively. Citation Format: Isis Y. Narvaez-Bandera, Ashley Lui, Eric Welsh, Dalia Ercan, Vanessa Rubio, Hayley Ackerman, Guohui Li, Lancia Darville, Min Liu, Bin Fang, Steven Eschrich, Brooke Fridley, John Koomen, Eric Haura, Gina M. DeNicola, Elsa Flores, Paul Stewart. Multi-omic landscape of squamous cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3495.

Comprehensive Characterization of Triterpene Saponins in Rhizoma Panacis Japonici by Offline Two-Dimensional Liquid Chromatography Coupled to Quadrupole Time-of-Flight Mass Spectrometry.

Rhizoma Panacis Japonici (RPJ) is an ancient herbal medicine from China that has long been employed for its medicinal benefits in relieving arthritis physical debility and diverse afflictions. The primary bioactive constituents found in RPJ are triterpene saponins, which exhibit numerous pharmacological actions, including anti-inflammatory, antioxidant, and immunomodulating effects. The present study established a straightforward and effective approach for characterizing triterpene saponins in RPJ. An offline HILIC × RP LC/QTOF-MS method was developed, along with a self-constructed in-house database containing 612 saponins reported in the Panax genus and 228 predicted metabolites. The approach achieved good chromatographic performance in isolating triterpene saponins of RPJ, with the HILIC column as the first dimension (1D) and the BEH C18 column as the second dimension (2D). The developed two-dimensional liquid chromatography system exhibited an orthogonality of 0.61 and a peak capacity of 1249. Detection was performed using a QTOF mass spectrometer in a data-independent manner (MSE) in a negative ion mode. Using the in-house database, the collected MS data were processed by an automatic workflow on UNIFI 1.8.2 software, which included data correction, matching of precursor and product ions, and peak annotation. In this study, 307 saponins were characterized from RPJ and 76 saponins were identified for the first time in Panax japonicus. This research not only enhances our understanding of the chemical characteristics of RPJ but also offers a simple and efficient method for analyzing the complex composition of herbal medicine.

Dereplication of calystegines in food plants and wild Solanum Brazilian fruits

Calystegines are potent glycosidase inhibitors with therapeutic potential and are constituents of food and feed with potential toxic effects. This study aims to target calystegines and other nitrogenous substances in food plants. Hydroalcoholic extracts from Solanum tuberosum, Ipomoea batatas, S. lycocarpum, and fruit from S. lycopersicum, S. aethiopicum, S. paniculatum, S. crinitum, and S. acanthodes were analyzed by liquid chromatography–tandem mass spectrometry (HPLC-MS/MS) using an acidic HILIC column. The dereplication approach included data processing using MZMine2, FBMN-GNPS, and structure elucidation and interpretation of the organized data. The calystegines A3, A5, B2, and C1 were identified, and several potential new calystegine analogues: three may correspond to new calystegines of the A-group, one glycosyl derivative of calystegine A3, and two glycosyl derivatives of the B-group. These findings help to direct the search for new calystegines. In addition, the dereplication approach enabled the annotation of 22 other nitrogen compounds.

Untargeted metabolomics of bladder tissue using liquid chromatography and quadrupole time-of-flight mass spectrometry for cancer biomarker detection

Bladder cancer (BC) ranks among the most common cancers globally, with an increasing occurrence, particularly in developed nations. Utilizing tissue metabolomics presents a promising strategy for identifying potential biomarkers for cancer detection. In this study, we utilized ultra-high-performance liquid chromatography coupled with ultra-high-resolution mass spectrometry (UHPLC-UHRMS), incorporating both C18-silica and HILIC columns, to comprehensively analyze both polar and non-polar metabolite profiles in tissue samples from 99 patients with bladder cancer. By utilizing an untargeted approach with external validation, we identified twenty-five tissue metabolites that hold promise as potential indicators of BC. Furthermore, twenty-five characteristic tissue metabolites that exhibit discriminatory potential across bladder cancer tumor grades, as well as thirty-nine metabolites that display correlations with tumor stages were presented. Receiver operating characteristics analysis demonstrated high predictive power for all types of metabolomics data, with area under the curve (AUC) values exceeding 0.966. Notably, this study represents the first report in which human bladder normal tissues adjacent to cancerous tissues were analyzed using UHPLC-UHRMS. These findings suggest that the metabolite markers identified in this investigation could serve as valuable tools for the detection and monitoring of bladder cancer stages and grades.

Development of targeted hydrophilic interaction liquid chromatography-tandem mass spectrometry method for acyl-Coenzyme A covering short- to long-chain species in a single analytical run

Acyl-CoAs play a significant role in numerous physiological and metabolic processes making it important to assess their concentration levels for evaluating metabolic health. Considering the important role of acyl-CoAs, it is crucial to develop an analytical method that can analyze these compounds. Due to the structural variations of acyl-CoAs, multiple analytical methods are often required for comprehensive analysis of these compounds, which increases complexity and the analysis time. In this study, we have developed a method using a zwitterionic HILIC column that enables the coverage of free CoA and short- to long-chain acyl-CoA species in one analytical run. Initially, we developed the method using an LC-QTOF instrument for the identification of acyl-CoA species and optimizing their chromatography. Later, a targeted HILIC-MS/MS method was created in scheduled multiple reaction monitoring mode using a QTRAP MS detector. The performance of the method was evaluated based on various parameters such as linearity, precision, recovery and matrix effect. This method was applied to identify the difference in acyl-CoA profiles in HepG2 cells cultured in different conditions. Our findings revealed an increase in levels of acetyl-CoA, medium- and long-chain acyl-CoA while a decrease in the profiles of free CoA in the starved state, indicating a clear alteration in the fatty acid oxidation process.