Liquid Chromatography-mass Spectrometry System Research Articles

Development of an advanced liquid chromatography–tandem mass spectrometry measurement system for simultaneous sphingolipid analysis

Mass spectrometry-based lipidomics approaches offer valuable tools for the detection and quantification of various lipid species, including sphingolipids. The present study aimed to develop a new method to simultaneously detect various sphingolipid species that applies to diverse biological samples. We developed and validated a measurement system by employing a single-column liquid chromatography-mass spectrometry system utilizing a normal-phase separation mode with positive ionization. The measurement system provided precision with a coefficient of variant below 20% for sphingolipids in all types of samples, and we observed good linearity in diluted serum samples. This system can measure the following sphingolipids: sphingosine 1-phosphate (S1P), sphingosine (Sph), dihydroS1P (dhS1P), dihydroSph (dhSph), ceramide 1-phosphate (Cer1P), hexosylceramide (HexCer), lactosylceramide (LacCer), dh-ceramide, deoxy-ceramide, deoxy-dh-ceramide, and sphingomyelin (SM). By measuring these sphingolipids in cell lysates where S1P lyase expression level was modulated, we could observe significant and dynamic modulations of sphingolipids in a comprehensive manner. Our newly established and validated measurement system can simultaneously measure many kinds of sphingolipids in biological samples. It holds great promise as a valuable tool for laboratory testing applications to detect overall modulations of sphingolipids, which have been proposed to be involved in pathogenesis processes in a series of elegant basic research studies.

On‐column trace‐level formation of N‐nitrosamine in a liquid chromatography–mass spectrometry analytical system

A model reaction between di‐n‐butylamine and sodium nitrite was studied to investigate trace‐level N‐nitrosamine formation. Liquid chromatography–mass spectrometry (LC–MS) analysis of kinetic time points from an in‐progress reaction showed a systematic offset in nitrosamine concentration between quenched and unquenched samples. By combining samples of amine and nitrite in the needle of the autosampler it was demonstrated that N‐nitrosamine was formed in the LC–MS system. Further experimentation indicated that nitrosation was occurring on‐column.

Dilute and shoot approach for toxicology testing.

Toxicology testing is performed in clinical settings, forensic settings, and for controlling doping. Drug screening is a toxicology test to determine if drugs are present in biological samples. The most common specimen type for drug testing is urine, as drugs and/or their metabolites are often more concentrated in the urine, extending the detection window of drugs. The dilute-and-shoot method is a simple procedure used in toxicology testing, where a sample is diluted before being directly injected into the liquid chromatography-mass spectrometry (LC-MS) system. This method is easy, quick, and cost-saving, and can be used for protein-poor liquid specimens such as urine. Thus, it is reasonable and attractive for busy toxicology laboratories to combine the dilute-and-shoot method with high-resolution hyphenated-MS for urine drug screening. This method has several disadvantages, including a suboptimal detection capability for certain analytes, as well as interference from co-eluting matrix components called matrix effects, in which co-eluting matrix molecules alter the ionization efficiency of the analyte molecules at the ionization source in LC-MS, altering (mostly reducing) the analyte detection capability. The matrix effect testing is essential for the validation of LC-MS-based assays. A reasonable approach to addressing these undesirable effects would be to minimize these components. The most straightforward approach is to reduce the amounts of matrix components by using a higher dilution of the specimen and a lower volume for specimen injection. Optimization of the chromatographic separation is another reasonable approach for reducing co-eluting matrix components with the analyte.

A cutting-edge approach based on UHPLC-MS to simultaneously investigate oxysterols and cholesterol precursors in biological samples: Validation in Huntington's disease mouse model

Brain is most cholesterol-rich organ in the body. Since cholesterol does not cross the blood brain barrier, its metabolism is provided in situ by astrocytes and neurons, and it is crucial for maintaining sterol levels and neuronal integrity and function. Recent studies have shown that the levels of cholesterol precursors and metabolites are lower in the brains of animal models of Huntington's disease (HD) while reduced levels of its catabolite are detected in the plasma of patients. In this study, we introduce a novel analytical method designed to fulfill the complex analytical requirements associated with cholesterol metabolites detection in neurodegenerative disorders. The method allows for the simultaneous quantification of a specific set of oxysterols along with cholesterol precursors in biological samples.The proposed method uses an Ultra-High-Performance Liquid Chromatography-Mass Spectrometry (UHPLC-MS) system operating in multiple reaction monitoring (MRM). Since sterols can be found in biological matrices in either free form or esterified to various fatty acids, a three-step extraction procedure was devised, consisting of alkaline hydrolysis, liquid-liquid extraction and final concentration omitting the need for a solid-phase extraction (SPE) step.The validated method achieved a detection limit of 10 ng/mL in plasma and 1 ng/mg in brain tissue, reaching a comparable sensitivity to previously published LC-MS and GC–MS methods. All target analytes were separated on a reverse-phase column employing a segmented gradient and a temperature ramp. This strategy enabled the elution and separation of all selected metabolites within a 30-minutes timeframe. This innovative approach was employed to quantify cholesterol metabolites in both plasma and brain samples from wild-type (WT) and R6/2 mice, a mouse model of HD. The results obtained from the sample analysis highlighted a significant reduction in desmosterol levels in the R6/2 brain at 12 weeks.In conclusion, the proposed method paves the way for further development of high-sensitive and reproducible protocols to comprehensively investigate simultaneous alterations in both cholesterol biosynthesis and catabolism in HD samples.

Application progress of on-line sample preparation techniques coupled with liquid chromatography-mass spectrometry system in the detection of food hazards

Application progress of on-line sample preparation techniques coupled with liquid chromatography-mass spectrometry system in the detection of food hazards

Untargeted-based metabolomics analysis and in vitro/in silico antiviral activity of extracts from Phyllanthus brasiliensis (Aubl.) Poir.

This study describes the molecular profile and the potential antiviral activity of extracts from Phyllanthus brasiliensis, a plant widely found in the Brazilian Amazon. The research aims to shed light on the potential use of this species as a natural antiviral agent. The extracts were analysed using liquid chromatography-mass spectrometry (LC-MS) system, a potent analytical technique to discover drug candidates. In the meantime, in vitro antiviral assays were performed against Mayaro, Oropouche, Chikungunya, and Zika viruses. In addition, the antiviral activity of annotated compounds was predicted by in silico methods. Overall, 44 compounds were annotated in this study. The results revealed that P. brasiliensis has a high content of fatty acids, flavones, flavan-3-ols, and lignans. Furthermore, in vitro assays revealed potent antiviral activity against different arboviruses, especially lignan-rich extracts against Zika virus (ZIKV), as follows: methanolic extract from bark (MEB) [effective concentration for 50% of the cells (EC50 ) = 0.80 μg/mL, selectivity index (SI) = 377.59], methanolic extract from the leaf (MEL) (EC50 = 0.84 μg/mL, SI = 297.62), and hydroalcoholic extract from the leaf (HEL) (EC50 = 1.36 μg/mL, SI = 735.29). These results were supported by interesting in silico prediction, where tuberculatin (a lignan) showed a high antiviral activity score. Phyllanthus brasiliensis extracts contain metabolites that could be a new kick-off point for the discovery of candidates for antiviral drug development, with lignans becoming a promising trend for further virology research.

System to screen and purify active ingredients from herbal medicines using hydrogel-modified human umbilical vein endothelial cell membrane chromatography coupled with semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry.

Analytical screening and validation systems based on a combination of cell membrane chromatography and two-dimensional chromatography-tandem mass spectrometry are incapable of providing prepared samples containing the active ingredients found in traditional Chinese medicine; therefore, these samples cannot be directly used in subsequent studies. In this study, a semi-preparative cell membrane chromatography column was developed using a hydrogel-modified carrier and human umbilical vein endothelial cells to optimize prepared conditions, such as hydrogel polymerization, cell fragmentation, and cell membrane volume. This increased the binding ratio of membrane protein and carrier to 15.79mg/g. The column was systematically evaluated using multitarget tyrosine kinase inhibitors that displayed good specificity and reproducibility. Subsequently, using the column coupled with a semi-preparative high-performance liquid chromatography-offline-high-performance liquid chromatography-mass spectrometry system, 15 active ingredients were screened and purified from Indigo naturalis, and five main components were identified: l-lysine, oxyresveratrol, tryptanthrin, isorhamnetin, and indirubin. Furthermore, the pharmacological effects of the ingredients were confirmed using cell proliferation and apoptosis assays. Results revealed potent proliferation-inhibiting and apoptosis-promoting abilities on human chronic myelogenous leukemic cells and human promyelocytic leukemic cells (p<0.001). Overall, the system presented screening and purification functions that could be used to prepare I. naturalis samples acting on the epidermal growth factor receptor and vascular endothelial cell growth factor.

Chemical profiling of Ziziphi spinosae semen using on-line comprehensive two-dimensional LC-MS based on a novel phthalic anhydride bonded stationary phase.

Ziziphi spinosae semen has been widely used to treat insomnia and anxiety. To profile its chemical components, an online comprehensive two-dimensional LC-MS was developed. In this two-dimensional LC system, a novel phthalic anhydride bonded stationary phase column was combined with a C18 column. As a result, this new stationary phase exhibited remarkable differences in separation selectivity from C18, achieving a good orthogonality of 83.3%. Moreover, this new stationary phase with weaker hydrophobicity than C18 realized solvent compatibility in the online configuration. Coupled with tandem MS, 154 compounds were identified, including 51 unreported compounds. Compared with one-dimensional LC-MS, this online two-dimensional LC-MS system exhibited a much higher resolving power in isomer separation. This work provided an effective separation and characterization method for the material basis of Ziziphi spinosae semen. This strategy provides ideas for the material basis research of other traditional Chinese medicines. This article is protected by copyright. All rights reserved.

Insights into the nitroaromatic compounds, formation, and light absorption contributing emissions from various geological maturity coals

Nitroaromatic compounds (NACs) are essential components of atmospheric organic aerosols. Coal combustion is a key source of atmospheric NACs. In this study, a triple-quadrupole liquid chromatography-mass spectrometry (LC-MS) system was used to identify ten individual NAC emitted in combustions of chunk coal and its briquette at different maturity levels. The Gaussian calculation was applied to quantify the absorption contribution of NACs to brown carbon (BrC). The emission factors (EFs) of total quantified NACs (ΣNACs) are 21.80–4429.55 μg/kg. 4-Nitrocatechol (4NC) is the most abundant NACs, accounting for 25.5–82.3 % of the ΣNACs and has the largest contribution to light absorption (0.34–29.23 %). The EFs for ΣNACs of chunk coal are 1.1–3.0 times those of its briquette, while the coal with volatile matter (VM) = 35.83 % shows the highest NAC emissions. The reaction pathway analysis demonstrates that NACs in briquette are generated through the pyrolysis of coal tar at an early stage of coal combustion, while volatile organic compounds (VOCs) that are emitted in chunk coal contribute greatly to the formations of NACs. The molecular properties analysis reveals that ΣNACs contribute 0.47–35.27 % to BrC light absorption. Anthracite coal (VM = 8.01 %) demonstrates the lowest light absorption coefficient (babs-365). Since bituminous coal (with VM = ~10 %–40 %) is popularly used for heating in rural China in winter, the results of this study could assist to evaluate the climate and environmental impacts on the NACs emission from coal combustion on a regional scale. Finally, the results highlighted that replacements of bituminous by clear fuel (such as chunk or briquette anthracite) could reduce NACs emission effectively.

Metabolomic changes in crown of alfalfa (Medicago sativa L.) during de-acclimation

Alfalfa is a high-quality forage legume species that is widely cultivated at high latitudes worldwide. However, a decrease in cold tolerance in early spring seriously affects regrowth and persistence of alfalfa. There has been limited research on the metabolomic changes that occur during de-acclimation. In this study, a liquid chromatography–mass spectrometry system was used to compare the metabolites in two alfalfa cultivars during a simulated overwintering treatment. In four pairwise comparisons, 367 differential metabolites were identified, of which 31 were annotated according to the Kyoto Encyclopedia of Genes and Genomes (KEGG) database. Many of these metabolites were peptides, carbohydrates, and lipids. At the subclass level, 17 major pathways were revealed to be significantly enriched (P < 0.05). The main differential metabolites included amino acids, peptides and analogs, carbohydrates, and glycerol phosphocholines. A metabolomic analysis showed that the up-regulation of unsaturated fatty acids and amino acids as well as the enhancement of the related metabolic pathways might be an effective strategy for increasing alfalfa cold tolerance. Furthermore, glycerophospholipid metabolism affects alfalfa cold tolerance in early spring. Study results provide new insights about the changes in alfalfa metabolites that occur during de-acclimation, with potential implications for the selection and breeding of cold-tolerant cultivars.

Distinct proteomic profiles in prefrontal subareas of elderly major depressive disorder and bipolar disorder patients

We investigated for the first time the proteomic profiles both in the dorsolateral prefrontal cortex (DLPFC) and anterior cingulate cortex (ACC) of major depressive disorder (MDD) and bipolar disorder (BD) patients. Cryostat sections of DLPFC and ACC of MDD and BD patients with their respective well-matched controls were used for study. Proteins were quantified by tandem mass tag and high-performance liquid chromatography-mass spectrometry system. Gene Ontology terms and functional cluster alteration were analyzed through bioinformatic analysis. Over 3000 proteins were accurately quantified, with more than 100 protein expressions identified as significantly changed in these two brain areas of MDD and BD patients as compared to their respective controls. These include OGDH, SDHA and COX5B in the DLPFC in MDD patients; PFN1, HSP90AA1 and PDCD6IP in the ACC of MDD patients; DBN1, DBNL and MYH9 in the DLPFC in BD patients. Impressively, depending on brain area and distinct diseases, the most notable change we found in the DLPFC of MDD was ‘suppressed energy metabolism’; in the ACC of MDD it was ‘suppressed tissue remodeling and suppressed immune response’; and in the DLPFC of BD it was differentiated ‘suppressed tissue remodeling and suppressed neuronal projection’. In summary, there are distinct proteomic changes in different brain areas of the same mood disorder, and in the same brain area between MDD and BD patients, which strengthens the distinct pathogeneses and thus treatment targets.

Methods for Quantifying the Metabolic Boundary Fluxes of Cell Cultures in Large Cohorts by High-Resolution Hydrophilic Liquid Chromatography Mass Spectrometry.

Metabolomics is a mainstream approach for investigating the metabolic underpinnings of complex biological phenomena and is increasingly being applied to large-scale studies involving hundreds or thousands of samples. Although metabolomics methods are robust in smaller-scale studies, they can be challenging to apply to larger cohorts due to the inherent variability of liquid chromatography mass spectrometry (LC-MS). Much of this difficulty results from the time-dependent changes in the LC-MS system, which affects both the qualitative and quantitative performances of the instrument. Herein, we introduce an analytical strategy for addressing this problem in large-scale microbial studies. Our approach quantifies microbial boundary fluxes using two zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) columns that are plumbed to enable offline column equilibration. Using this strategy, we show that over 397 common metabolites can be resolved in 4.5 min per sample and that metabolites can be quantified with a median coefficient of variation of 0.127 across 1100 technical replicates. We illustrate the utility of this strategy via an analysis of 960 strains of Staphylococcus aureus isolated from bloodstream infections. These data capture the diversity of metabolic phenotypes observed in clinical isolates and provide an example of how large-scale investigations can leverage our novel analytical strategy.

Recent advances in LC-MS-based metabolomics for clinical biomarker discovery.

The employment of liquid chromatography-mass spectrometry (LC-MS) untargeted and targeted metabolomics has led to the discovery of novel biomarkers and improved the understanding of various disease mechanisms. Numerous strategies have been reported to expand the metabolite coverage in LC-MS-untargeted and targeted metabolomics. To improve the sensitivity of low-abundance or poor-ionized metabolites for reducing the amount of clinical sample, chemical derivatization methods are used to target different functional groups. Proper sample preparation is beneficial for reducing the matrix effect, maintaining the stability of the LC-MS system, and increasing the metabolite coverage. Machine learning has recently been integrated into the workflow of LC-MS metabolomics to accelerate metabolite identification and data-processing automation, and increase the accuracy of disease classification and clinical outcome prediction. Due to the rapidly growing utility of LC-MS metabolomics in discovering disease markers, this review will address the recent advances in the field and offer perspectives on various strategies for expanding metabolite coverage, chemical derivatization, sample preparation, clinical disease markers, and machining learning for disease modeling.

Postmortem forensic toxicology cases: A retrospective review from Milan, Italy.

We are presenting a study on 136 cases performed in a 2‐year period (2018–2019) at the Bureau of Legal Medicine of the University of Milan for which toxicological analyses were requested and we are making a detailed interpretation of clinical records and discussing toxicological results from each case included in the study. Total number of autopsies was 1323 and in 10.3% of the cases, toxicological analyses were requested to obtain further information. Analyses were assessed with High‐Performance Liquid Chromatography‐Mass Spectrometry system and Gas Chromatography–Mass Spectrometry analyses. Additionally, Blood Alcohol Concentration and detection of volatile substances were obtained with Head Space‐Gas Chromatography–Mass Spectrometry system. From these analyses, 101 cases out of 136 provided positive results (74.3%). Main substances detected were cocaine, diazepam, morphine, and ethanol. The most representative profiles of individuals that emerged from this study were: a Caucasian male, age 41–50, that died for cocaine acute intoxication or was killed; a Caucasian male or female with a range‐of‐age of 31–50 deceased for simple suicide caused by acute intoxication or by complex suicide caused by acute intoxication and suffocation; and finally, a Caucasian male with a range‐of‐age 21–40 that died in a car accident without any toxicological evidence. From the results, acute intoxication at the time of death was confirmed in 54 cases and in 57 cases the toxicological analyses helped in the determination of the cause and manner of death. From this study, the importance of toxicological data among forensic sciences is confirmed.

Food glycomics: Dealing with unexpected degradation of oligosaccharides during sample preparation and analysis.

This study reveals that unexpected degradation of food oligosaccharides can occur during conventional glycomics workflows, including sample preparation and analysis by liquid chromatography-mass spectrometry (LC-MS). With the present investigation, we aim to alert the scientific community of the susceptibility of specific glycosidic linkages to degradation induced by heat and acid. Key standard oligosaccharides representing the major types found in foods (3'-sialyllactose and 6'-sialyl-N-acetyllactosamine for milk, raffinose and stachyose for legumes) were selected as model systems and underwent each of the following treatments independently: (1) labeled with the derivatizing agent 1-aminopyrene-3,6,8-trisulfonic (APTS) (followed by analysis with a capillary electrophoresis system coupled with a fluorescence detector), (2) dried from an acetonitrile-water mixture containing 0.1% trifluoroacetic acid, and (3) injected into an LC-MS system. We demonstrated that both raffinose and stachyose degraded during APTS-labeling by the acid in the labeling reagents. We also discovered that during centrifugal evaporation at 37 °C, all of the four nonderivatized oligosaccharides tested were partially degraded. Additionally, when the LC-MS eluent contained 0.1% formic acid, 3'-sialyllactose, raffinose, and stachyose underwent extensive in-source fragmentation during analysis. Lastly, we identified a simple strategy that can reduce the probability of incorrect oligosaccharide identification resulting from extensive in-source fragmentation.

Template-directed synthesis of pomegranate-shaped zinc oxide@zeolitic imidazolate framework for visible light photocatalytic degradation of tetracycline

The development of photocatalysts for efficient tetracycline (TC) degradation under visible light is urgently needed yet remains a great challenge. Most semiconductor photocatalysts with low specific surface area are easy to agglomerate in solution and unfavorable for enriching pollutants. Herein, we present the preparation of pomegranate-shaped zinc oxide@zeolitic imidazolate framework (ZnO@ZIF-8) by in situ growth of ZIF-8 on a petal-shaped ZnO template that enhances the adsorption and photocatalytic degradation of TC. ZnO@ZIF-8 exhibits an excellent photostability and a TC photodegradation efficiency of 91% under visible light (λ > 420 nm) in 50 min at room temperature, which can be recycled over five times without any loss of activity. Moreover, the plausible photocatalysis reaction mechanism and the degradation intermediates are elucidated with the aid of three-dimensional excitation−emission matrix spectra and liquid chromatography-mass spectrometry system. This study offers new insights into the design of antibiotic degradation photocatalysts and the development of photocatalysts with broad-spectrum responses for efficient TC elimination.

Multiplex Lysosomal Enzyme Activity Assay on Dried Blood Spots Using Tandem Mass Spectrometry.

Deficiencies of the enzymes in lysosomes result in the accumulation of undegraded materials and subsequently cellular dysfunction. Early identification of deficiencies can lead to better clinical outcomes before irreversible organ and tissue damages occur. In this chapter, lysosomal enzymes are extracted from dried blood spots and incubated with the commercialized and multiplexed enzyme cocktail containing corresponding substrates and internal standards. After incubation, the enzymatic reactions are quenched, and the mixtures of the reaction products are prepared using liquid/liquid extractions. Multiple enzymes are quantified simultaneously using selected ion monitoring on liquid chromatography-mass spectrometry (LC-MS/MS) system.

Changes in Meibum Lipid Composition With Ocular Demodex Infestation.

PurposeThe purpose of this study was to understand the impact of Demodex infection in the lipid component of meibum in patients.MethodsThe meibum samples were collected from four groups of subjects: (1) Demodex-negative with non-MGD (D−M−; n = 10); (2) Demodex-positive with non-MGD (D+M−; n = 10); (3) Demodex-negative with MGD (D−M+; n = 10); and (4) Demodex-positive with MGD (D+M+; n = 10). A liquid chromatography–mass spectrometry (LC-MS) system consisting of ultra-performance liquid chromatography and a Q Exactive high-resolution mass spectrometer was used for lipids separation and detection.ResultsCompared with the D−M− group, the D+M− group had lower levels of phosphatidylcholines (PCs) and lysophosphatidylcholines (LPCs) and higher levels of phosphatidylethanolamines (PEs). Compared with the D−M+ group, the levels of sphingomyelins (SMs) and PCs in the D+M+ group were decreased, whereas the levels of (O-acyl)-ω-hydroxy fatty acids (OAHFAs), ceramides (CERs), LPCs, and diacylglycerols (DGs) were significantly increased. Triacylglycerols (TGs), DGs, CERs, and OAHFAs were decreased in D−M+ group, whereas levels of PEs, phosphatidylinositols, and phosphatidylglycerols were increased in meibum obtained from the D−M+ group compared with those in the D−M− group. TGs, SMs, CERs, and PEs were decreased in the D+M+ group, whereas levels of LPCs, LPEs, PCs, and PEs were increased in meibum from the D−M+ group compared with those in the D+M− group.ConclusionsTo the best of our knowledge, this is the first study to assess the changes in meibum from patients with ocular Demodex infestation. The significant increase of OAHFAs in the Demodex-positive group suggest that OAHFAs may be associated with the progress of ocular Demodex infections.Translational RelevanceOAHFAs could be a potential new therapeutic target for ocular Demodex infestation.

Native mass spectrometry-based metabolomics identifies metal-binding compounds.

Although metals are essential for the molecular machineries of life, systematic methods for discovering metal-small molecule complexes from biological samples are limited. Here, we describe a two-step native electrospray ionization-mass spectrometry method, in which post-column pH adjustment and metal infusion are combined with ion identity molecular networking, a rule-based data analysis workflow. This method enabled the identification of metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion species with the same chromatographic profiles. As this native electrospray metabolomics approach is suited to the use of any liquid chromatography-mass spectrometry system to explore the binding of any metal, this method has the potential to become an essential strategy for elucidating metal-binding molecules in biology.

Engineering an integrated system with a high pressure polymeric microfluidic chip coupled to liquid chromatography-mass spectrometry (LC-MS) for the analysis of abused drugs

A microfluidic chip coupled to a liquid chromatography-mass spectrometry (LC-MS) system is a powerful tool to determine components of interest in tracing biological samples. However, producing a reusable and robust chip having chromatographic separation for LC-MS systems remains analytically challenging due to the high back pressure generally occurring when microfluidic channels are packed particles. In this study, a one-piece and high-pressure-resistant polymeric microfluidic chip was fabricated by commercial stereolithographic 3D printing with an inner diameter of 1 mm, packed with 5 µm diameter fluorophenyl propyl particles, and coupled to a regular LC-MS. The maximum pressure occurred up to 134 bar with a variation of 1 bar when using 70% methanol at a flow rate of 0.035 mL min-1. Ten drugs and two drug metabolites were analyzed in 15 min under continuous pressure change by LC-MS. The limit of detection was between 2 and 20 ng mL-1 in human urine. The coefficient of determination (R2) was > 0.991. The shift of retention time of each peak was maintained at < 2%. After a chip was eluted with a high organic solvent composition (90% methanol/water and 90% acetonitrile/water mixture) and a high back pressure for continuously 45 h, the cross-sectional areas were investigated and results showed that the microfluidic chip remained intact. The overall experiment results showed that this platform had significant reproducibility, robustness and stability without contamination, demonstrating that this 3D printing microfluidic chip coupled with LC-MS is an efficient and precise tool for bioanalysis.