Discovery of potential therapeutic targets for non-small cell lung cancer using high-throughput metabolomics analysis based on liquid chromatography coupled with tandem mass spectrometry

Lung cancer is a severe health problem and threatens a patient''s quality of life. The metabolites present in biological systems are expected to be key mediators and the changes in these metabolites play an important role in promoting health. Metabolomics can unravel the global metabolic changes and identify significant biological pathways involved in disease development. However, the role of metabolites in lung cancer is still largely unknown. In the present study, we developed a liquid chromatography coupled with tandem mass spectrometry method for biomarker discovery and identification of non-small cell lung cancer (NSCLC) from metabolomics data sets and aimed to investigate the metabolic profiles of NSCLC samples to identify potential disease biomarkers and to reveal the pathological mechanism. After cell metabolite extraction, the metabolic changes in NSCLC cells were characterized and targeted metabolite analysis was adopted to offer a novel opportunity to probe into the relationship between differentially regulated cell metabolites and NSCLC. Quantitative analysis of key enzymes in the disturbed pathways by proteomics was employed to verify metabolomic pathway changes. A total of 13 specific biomarkers were identified in NSCLC cells related with metabolic disturbance of NSCLC morbidity, which were involved in 4 vital pathways, namely glycine, serine and threonine metabolism, aminoacyl-tRNA biosynthesis, tyrosine metabolism and sphingolipid metabolism. The proteomics analysis illustrated the obvious fluctuation of the expression of the key enzymes in these pathways, including the downregulation of 3-phosphoglycerate dehydrogenase, phosphoserine phosphatase, tyrosinase and argininosuccinic acid catenase. NSCLC occurrence is mainly related to amino acid and fatty acid metabolic alteration. These findings highlight that the metabolome can provide information on the molecular profiles of cells, which can aid in investigating the metabolite changes to reveal the pathological mechanism.

High-throughput metabolomics can discover potential therapeutic targets for non-small cell lung cancer.     

High-throughput system metabolomics method reveals new mechanistic insights of chlorogenic acid by using liquid chromatography coupled to high resolution mass spectrometry

It has increasingly been recognized that metabolism is highly interconnected with disease, and system metabolomics studies have aimed to discover metabolic biomarkers and analyze the pathways of metabolome phenotypes. To better understand the metabolic alteration related with disease, a urine metabolic profile using a high-throughput system metabolomics technology approach was applied to probe the underlying molecular mechanisms of alcohol-induced liver injury and the therapeutic effects of chlorogenic acid (CA). In this study, endogenous low-molecular-weight metabolites were characterized using liquid chromatography coupled with mass spectrometry (LC-MS). The acquired data was parsed by principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) to identify potential biomarkers. A total of 19 biomarkers were identified in a model of alcohol-induced liver injury rats, and it was found that chlorogenic acid had a regulatory effect on 14 of them, associated with multiple metabolic pathways. Comprehensive pathway analysis suggests that CA has the ability to regulate abnormal metabolic states. In addition, accessory examinations such as biochemical analysis and histopathological observations were also performed that showed similar results. As a natural product agent against ethanol-induced liver injury, CA was validated in the rebalancing of a wide range of metabolic disorders. High-throughput system metabolomics represents a powerful approach for revealing new mechanistic insights of natural products.

It has increasingly been recognized that metabolism is highly interconnected with disease, and system metabolomics studies have aimed to discover metabolic biomarkers and analyze the pathways of metabolome phenotypes.     

Exploring metabolic biomarkers and regulation pathways of acute pancreatitis using ultra-performance liquid chromatography combined with a mass spectrometry-based metabolomics strategy

Acute pancreatitis (AP), as a common kind of pancreas-based inflammatory disease, is accompanied by a serious and abnormal metabolism. However, the specific metabolic process of AP is still unclear. Novel and effective drugs against acute pancreatitis are urgently required. To explore the metabolic biomarkers and regulation pathways of acute pancreatitis, ultra-performance liquid chromatography (UPLC) combined with a mass spectrometry (MS)-based metabolomics strategy was used. Sixteen male adult Sprague-Dawley rats were divided into two groups: a sham operation group (SO) and an AP model group. The AP animal model was induced via the retrograde ductal infusion of 3.5% sodium taurocholate, and rats in the SO group were infused with 0.9% saline. After serum sample collection and sacrifice, a metabolomics strategy based on UPLC-MS was used to detect serum metabolites and metabolic pathways by comparing the SO and AP model groups through full-scan analysis. A total of 19 metabolites were detected in the serum for highlighting the differences between the two groups: l-arabitol, citric acid, isocitric acid, l-phenylalanine, l-tyrosine, dihydroxyacetone, l-valine, succinic acid, 3-hydroxybutyric acid, uric acid, acetylglycine, palmitic amide, homocysteine, d-glutamine, l-arginine, arachidonic acid, N-acetylserotonin, (R)-3-hydroxy-hexadecanoic acid, and d-mannose. Six crucial metabolic pathways, phenylalanine, tyrosine and tryptophan biosynthesis, arachidonic acid metabolism, glyoxylate and dicarboxylate metabolism and the citrate cycle, were involved; these have potential to become novel targets for the treatment of AP. The ingenuity pathway analysis (IPA) platform is used to gain insights into the metabolic targets in the system, referring to development disorders, cell-to-cell signaling and interactions, cellular assembly and organization, cell compromise, cell growth and proliferation, carbohydrate metabolism and others. It is suggested that UPLC-MS-based metabolomics is capable of accurately depicting the pathological mechanisms of acute pancreatitis, which can drive new drug development.

Acute pancreatitis (AP), as a common kind of pancreas-based inflammatory disease, is accompanied by a serious and abnormal metabolism.     

Applications of liquid chromatography coupled to mass spectrometry-based metabolomics in clinical chemistry and toxicology: A review

The metabolome is the set of small molecular mass organic compounds found in a given biological media. It includes all organic substances naturally occurring from the metabolism of the studied living organism, except biological polymers, but also xenobiotics and their biotransformation products. The metabolic fingerprints of biofluids obtained by mass spectrometry (MS) or nuclear magnetic resonance (NMR)-based methods contain a few hundreds to thousands of signals related to both genetic and environmental contributions. Metabolomics, which refers to the untargeted quantitative or semi-quantitative analysis of the metabolome, is a promising tool for biomarker discovery. Although proof-of-concept studies by metabolomics-based approaches in the field of toxicology and clinical chemistry have initially been performed using NMR, the use of liquid chromatography hyphenated to mass spectrometry (LC/MS) has increased over the recent years, providing complementary results to those obtained with other approaches. This paper reviews and comments the input of LC/MS in this field. We describe here the overall process of analysis, review some seminal papers in the field and discuss the perspectives of metabolomics for the biomonitoring of exposure and diagnosis of diseases.     

Pharmacokinetic profile and metabolite identification of bornyl caffeate and caffeic acid in rats by high performance liquid chromatography coupled with mass spectrometry

Bornyl caffeate was initially discovered as a bioactive compound in medicinal plants. Despite the promising pharmacological activities including anti-tumor and antibacterial activities, the pharmacokinetics of the compound remain open. This work developed a high performance liquid chromatography-tandem mass spectrometric method for the determination of bornyl caffeate and caffeic acid (major metabolite and a main unit of bornyl caffeate) in vivo. Successful application of the method included identification of its metabolites and investigation on the drug pharmacokinetics. A total of 30 compounds were identified as the metabolites of bornyl caffeate in rats. We attributed these metabolites to phase I metabolic routes of reduction, oxidation, hydrolysis and phase II metabolic reactions of glucuronidation, sulfation, O-methylation and glycine. Glucuronidation, sulfation, O-methylation and reduction were the main metabolic pathways of bornyl caffeate. The method presented a linear range of 1–4000 ng mL⁻¹. The pharmacokinetic profile of bornyl caffeate was found to be a three compartment open model, while caffeic acid fitted to a two compartment open model when it was administered alone or served as the main metabolite of bornyl caffeate. The time to peak concentration (T_max) and the maximum plasma concentration (C_max) of bornyl caffeate were 0.53 h and 409.33 ng mL⁻¹. Compared with original caffeic acid, the compound displayed an increased half-life of elimination (T_1/2β), area under the concentration time curve from 0 to t (AUC_0–t) and area under the concentration time curve from 0 to ∞ (AUC_0–∞), a decreased half-life of absorption (T_1/2α) and an identical C_max. Taking together, we concluded that bornyl caffeate is able to rapidly initiate therapeutic effect and last for a relatively long time in rats; metabolic pathways of O-methylation and reduction is key to interpret the mechanism and toxicity of bornyl caffeate.

We revealed the metabolic profile of bornyl caffeate by HPLC-Q-TOF/MS, and then simultaneously examined the pharmacokinetics of bornyl caffeate and CA after administration of a single dose of bornyl caffeate by HPLC ion trap MS.     

Nontargeted SWATH acquisition mode for metabolites identification of osthole in rats using ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry

Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in Cnidium monnieri (L.) Cuss. and possesses excellent pharmacological activities, including anticancer, anti-apoptosis and neuroprotection. In this study, a rapid and reliable method based on ultra-high-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF-MS) and MetabolitePilot2.0™ software with principal component variable grouping (PCVG) filtering was developed to observe probable metabolites of OST firstly. The high resolution mass data were acquired by data-independent acquisition mode (DIA), i.e., sequential window acquisition of all theoretical fragmentation spectra (SWATH), which could significantly improved the hit rate of low-level and trace metabolites. A novel data processing method ‘key product ions (KPIs)’ were employed for metabolites rapid hunting and identification as an assistant tool. A total of 72 metabolites of OST were detected in vitro and in vivo, including 39 metabolites in rat liver microsomes (RLMs), 20 metabolites in plasma, 32 metabolites in bile, 32 metabolites in urine and 37 metabolites in feces. The results showed that mono-oxidation, demethylation, dehydrogenation, sulfate conjugation and glucuronide conjugation were major metabolic reactions of OST. More significant, oxydrolysis, 3,4-epoxide-aldehylation, phosphorylation, S-cysteine conjugation and N-acetylcysteine conjugation were considered as unique metabolic pathways of OST, and phosphorylation, S-cysteine conjugation and N-acetylcysteine conjugation reactions were characterized in rat biological samples for the first time. Preparation of active metabolites will be greatly helpful in elucidating the potential biological mechanism of OST, and the proposed metabolic pathways of it might provide further understanding of the safety and efficacy of simple coumarins.

Osthole (OST), 7-methoxy-8-isopentenoxycoumarin, is the characteristic constituent found in Cnidium monnieri (L.) Cuss. and possesses excellent pharmacological activities, including anticancer, anti-apoptosis and neuroprotection.     

Biomarker discovery in biological specimens (plasma, hair, liver and kidney) of diabetic mice based upon metabolite profiling using ultra-performance liquid chromatography with electrospray ionization time-of-flight mass spectrometry

BackgroundThe number of diabetic patients has recently been increasing worldwide. Diabetes is a multifactorial disorder based on environmental factors and genetic background. In many cases, diabetes is asymptomatic for a long period and the patient is not aware of the disease. Therefore, the potential biomarker(s), leading to the early detection and/or prevention of diabetes mellitus, are strongly required. However, the diagnosis of the prediabetic state in humans is a very difficult issue, because the lifestyle is variable in each person. Although the development of a diagnosis method in humans is the goal of our research, the extraction and structural identification of biomarker candidates in several biological specimens (i.e., plasma, hair, liver and kidney) of ddY strain mice, which undergo naturally occurring diabetes along with aging, were carried out based upon a metabolite profiling study.MethodsThe low-molecular-mass compounds including metabolites in the biological specimens of diabetic mice (ddY-H) and normal mice (ddY-L) were globally separated by ultra-performance liquid chromatography (UPLC) using different reversed-phase columns (i.e., T3-C18 and HS-F5) and detected by electrospray ionization time-of-flight mass spectrometry (ESI-TOF-MS). The biomarker candidates related to diabetes mellitus were extracted from a multivariate statistical analysis, such as an orthogonal partial least-squares-discriminant analysis (OPLS-DA), followed by a database search, such as ChemSpider, KEGG and HMDB.ResultsMany metabolites and unknown compounds in each biological specimen were detected as the biomarker candidates related to diabetic mellitus. Among them, the elucidation of the chemical structures of several possible metabolites, including more than two biological specimens, was carried out along with the comparison of the tandem MS/MS analyses using authentic compounds. One metabolite was clearly identified as N-acetyl-l-leucine based upon the MS/MS spectra and the retention time on the chromatograms.ConclusionsN-acetyl-l-leucine is an endogenous compound included in all biological specimens (plasma, hair, liver and kidney). Therefore, this metabolite appears to be a potential biomarker candidate related to diabetes. Although the structures of other biomarker candidates have still not yet determined, the present approach based upon a metabolite profiling study using UPLC-ESI-TOF-MS could be helpful for understanding the abnormal state of various diseases.     

Comprehensive profiling of Platycodonis radix in different growing regions using liquid chromatography coupled with mass spectrometry: from metabolome and lipidome aspects

Platycodon grandiflorus (Jacq.) A. DC. is widely cultivated across the south and north of China. Its root, Platycodonis radix, is commonly used as a vegetable, functional food, and traditional herbal medicine with various biological benefits. It is critical to fully clarify the chemical composition of Platycodonis radix for the sake of the food industry and traditional herb markets. In this study, a strategy of metabolome and lipidome profiling based on ultra-high performance liquid chromatography coupled to ion mobility-quadrupole time of flight mass spectrometry (UPLC-IM-QTOF-MS) was developed to reveal the overall chemical composition of Platycodonis radix. IN particular, comprehensive lipidome profiling was first performed for Platycodonis radix, in which 170 lipid molecular species including 55.9% glycerophospholipids, 31.2% glycerolipids, and 12.9% sphingolipids were identified. Platycodonis radix from two major production regions in China, Inner Mongolia and Anhui province, were collected and analyzed by the MS based approach combined with multivariate statistical analysis from both the metabolome and lipidome aspects. This study threw focus on the profiling investigations of Platycodonis radix from different growing regions and provided new potential in the lipidome analysis of medicinal food.

Lipidome and metabolome profiling investigations of Platycodonis radix in different growing regions.     

Metabolomic estimation of the diagnosis of hepatocellular carcinoma based on ultrahigh performance liquid chromatography coupled with time-of-flight mass spectrometry

Metabolomics has been shown to be an effective tool for biomarker screening and pathway characterization and disease diagnosis. Metabolic characteristics of hepatocellular carcinoma (HCC) may enable the discovery of novel biomarkers for its diagnosis. In this work, metabolomics was used to investigate metabolic alterations of HCC patients. Plasma samples from HCC patients and age-matched healthy controls were investigated using high resolution ultrahigh performance liquid chromatography-mass spectrometry and metabolic differences were analyzed using pattern recognition methods. 23 distinguishable metabolites were identified. The altered metabolic pathways were associated with arginine and proline metabolism, glycine, serine and threonine metabolism, steroid hormone biosynthesis, starch and sucrose metabolism, etc. To demonstrate the utility of plasma biomarkers for the diagnosis of HCC, five metabolites comprising deoxycholic acid 3-glucuronide, 6-hydroxymelatonin glucuronide, 4-methoxycinnamic acid, 11b-hydroxyprogesterone and 4-hydroxyretinoic acid were selected as candidate biomarkers. These metabolites that contributed to the combined model could significantly increase the diagnostic performance of HCC. It has proved to be a powerful tool in the discovery of new biomarkers for disease detection and suggest that panels of metabolites may be valuable to translate our findings to clinically useful diagnostic tests.

Metabolomics has been shown to be an effective tool for biomarker screening and pathway characterization and disease diagnosis.     

Identification of the cytochrome P450 enzymes involved in the oxidative metabolism of trantinterol using ultra high-performance liquid chromatography coupled with tandem mass spectrometry

Trantinterol is a novel β₂-adrenoceptor agonist used for the treatment of asthma. This study aimed to identify the cytochrome P450 enzymes responsible for the metabolism of trantinterol to form 4-hydroxylamine trantinterol (M1) and tert-butyl hydroxylated trantinterol (M2), which was achieved using the chemical inhibition study, followed by the metabolism study of trantinterol in a panel of recombinant CYPs, as well as the kinetic study with the appropriate cDNA-expressed P450 enzymes. A highly selective and sensitive ultra high-performance liquid chromatography tandem mass spectrometry method was developed and validated for the simultaneous determination of M1 and M2. The inhibition study suggested that CYP2C19 and CYP3A4/5 were involved in the formation of M1 and M2, and CYP2D6 only contributed to the formation of M1. Assays with cDNA-expressed CYP enzymes further showed that the relative contributions of P450 isoforms were 2C19 > 3A4 > 2D6 > 2E1 for the formation of M1, and 3A4 > 2C19 > 2D6 for the formation of M2. The enzyme kinetic analysis was then performed in CYP2C19, CYP2D6 and CYP3A4. The kinetic parameters were determined and normalized with respect to the human hepatic microsomal P450 isoform concentrations. All the results support the conclusion that CYP3A4 and CYP2C19 are the major enzymes responsible for formation of M1 and M2, while CYP2D6 and CYP2E1 also engaged to a lesser degree. The results imply that potential drug–drug interactions may be noticed when trantinterol is used with CYP2C19 and CYP3A4 inducers or inhibitors, and we should pay attention to this phenomenon in clinical study.

The first report on the characterization of the main CYP450 enzymes and the kinetic study involved in trantinterol metabolism.     

Simultaneous quantification of five biomarkers in ethanolic extract of Cassia occidentalis Linn. stem using liquid chromatography tandem mass spectrometry: application to its pharmacokinetic studies

Cassia occidentalis L. stem extract is used as a purgative, febrifuge, and diuretic, and in the treatment of flu, fever, fracture and bone diseases. Pharmacological studies prove the osteogenic and antiresorptive effects of Cassia occidentalis L. ethanolic extract (COEE), which may be due to apigenin, apigenin-6-C-glucopyranoside, luteolin, 3′,4′,7-trihydroxyflavone and emodin. The objectives of this study was to develop a selective and sensitive LC-MS/MS method and validate for the simultaneous determination of the above five biomarkers in rat plasma after oral administration of COEE at a dose of 500 mg kg⁻¹. The analytes were separated on a Phenomenex Luna C18 column (4.6 × 150 mm, 3.0 μm) with an isocratic mobile phase consisting of methanol-10 mM ammonium acetate buffer (95 : 05, v/v). Run time was for 5.5 min with LLOQ of 1 ng mL⁻¹ for all the analytes. The mass spectrometer was operating in negative ionization mode for quantification of the analytes. The calibration curves were linear (r² > 0.99) for all the analytes. The intra- and inter-day precisions were less than 8.17% and the relative error was between −8.57% and 7.28%. Analytes were rapidly absorbed in the oral pharmacokinetic study. The biomarkers were stable in simulated gastric and intestinal fluids but underwent metabolism in rat liver microsomes. This is the first report on in vivo oral pharmacokinetics and in vitro stability studies of osteogenic compounds present in COEE. These results will be helpful for further understanding of pharmacodynamic behaviour of COEE and the bioanalytical method will be useful for further preclinical/clinical trials.

Liquid chromatography tandem mass spectrometry was used to develop a bioanalytical method for simultaneous quantification of five biomarkers of Cassia occidentalis L. ethanolic extract to use in oral pharmacokinetics and in vitro stability studies.     

A validated assay for the quantitative analysis of tranexamic acid in human serum by liquid chromatography coupled with electrospray ionization mass spectrometry

BackgroundTranexamic acid is a synthetic lysine analog used for management of bleeding disorders. The objective of this study was first to develop a method for measurement of tranexamic acid in human serum using liquid chromatography coupled to ion-trap mass spectrometry (LC-MS/MS), and then to validate it throughout a wide range of concentrations allowing quantification in patients receiving tranexamic acid infusion during surgery.MethodsSerum samples (100 µL) were subjected to protein precipitation with perchloric acid, and after pH adjustment, tranexamic acid and internal standard were separated on a C₁₈ column and isocratically eluted using a mobile phase constituted of formate buffer/acetonitrile (95:5, v/v). Tranexamic acid was ionized by electrospray in positive mode. Parent [M + H]⁺ ions were m/z 158.0 for tranexamic acid and m/z 144.0 for IS. The most intense product ion of tranexamic acid (m/z 122.7) and IS (m/z 126.0) were used for quantification.ResultsThe assay was accurate and precise over the range of 1.0 (lower limit of quantification) to 200.0 µg/mL (upper limit of quantification), and has been successfully applied to study the clinical pharmacokinetics in two volunteers undergoing cardiac surgery.ConclusionA reliable method for quantification of tranexamic acid for analysis in clinical studies was obtained.     

Flow injection analysis vs. ultra high performance liquid chromatography coupled with tandem mass spectrometry for determination of imatinib in human plasma

BackgroundThe aim of this study was to develop, validate and compare flow injection analysis (FIA) and ultra-high-performance liquid chromatography (LC)/tandem mass spectrometry methods for the determination of imatinib in plasma from patients with chronic myeloid leukemia.MethodsThe plasma for analysis by both methods was deproteinated by methanol containing d8-imatinib. The separation was achieved on a 1.7 μm C18 column with a linear gradient (4 mM ammonium formiate and acetonitrile, pH 3.2). FIA was performed at flow rate of 0.03 mL/min (0.1% formic acid in methanol). Multiple reaction monitoring mode on the tandem mass spectrometer (API 4000, AB Sciex) in positive ESI were used for detection.ResultsThe total analysis times were 3.2 (LC) and 0.75 min (FIA). Both methods were successfully validated and applied to the plasma patients samples. The limits of quantification were 4.1 and 30.8 ng/mL; imprecisions were less than 5.7% and recovery ranged between 93 and 105%, for the LC and FIA, respectively. The methods revealed an agreement with a mean difference of 1.46 ng/mL (SD 28.95 ng/mL).ConclusionsThe high-throughput methods that were developed are suitable for the therapeutic drug monitoring of imatinib in plasma. They can be used in routine clinical practice.     

A rapid liquid chromatography-tandem mass spectrometry analysis of whole blood sirolimus using turbulent flow technology for online extraction

BACKGROUND: Sirolimus is an immunosuppressant used in solid organ transplantation. Due to variable individual pharmacokinetics and narrow therapeutic ranges, therapeutic drug monitoring (TDM) is critical to the success of post-transplantation patient care. We developed a rapid method quantifying whole blood sirolimus using liquid chromatography-tandem mass spectrometry (LC-MS/MS) with an automated online extraction technology. METHODS: Whole blood (100 microL) was mixed with a precipitation solution containing internal standard (32-desmethoxyrapamycin) and centrifuged at 15,634 x g for 10 min. The supernatant (50 microL) was injected onto a turbulent flow preparatory column and then a C18 analytical column. The mass spectrometer was set for positive electrospray to monitor the ammonium adducts. RESULTS: Analytical time was 4 min/injection. Inter- and intra-assay variation coefficients across three concentration levels ranged from 2.3% to 7.4%. The method was linear from 1.0 to 100.0 ng/mL with an accuracy of 93.3%-100.0%. No carryover was detected from samples at 313.6 ng/mL. There was no obvious ion suppression from patient samples or interference from other commonly used immunosuppressants. Good correlation with an in-house commercial LC-MS was observed. CONCLUSIONS: The LC-MS/MS method coupled with turbulent flow technology is rapid and efficient in TDM of whole blood sirolimus.