Hydrophilic Interaction Liquid Chromatography-tandem Mass Research Articles

Determination of adenosine and its modifications in urine and plasma from breast cancer patients by hydrophilic interaction liquid chromatography-tandem mass spectrometry

RNA modifications have been revealed to be essential in many biological activities, and their disorders are associated with various human diseases, including cancers. 2′-O-methyladenosine (Am), N1-methyladenosine (m1A), N6-methyladenosine (m6A), N6,2′-O-dimethyladenosine (m6Am) and N6,N6-dimethyladenosine (m62A) are important adenosine (A) modifications. The noninvasive collection of urine samples and the diverse contents of metabolites in plasma make them favored biofluids for biomarkers discovery. In this work, we established a hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method to quantify these six nucleosides in urine and plasma of healthy controls and breast cancer (BC) patients. The limit of detection (LOD) for A, Am, m1A, m6A, m6Am, and m62A were 0.0025, 0.01, 0.05, 0.005, 0.005, and 0.005 nM. The results showed that the concentrations of Am, m6A, and m6Am were increased, whereas m1A was decreased in the urine of BC patients compared with the healthy controls. We also found that the level ratios of m1A/A, m6A/A, and m6Am/A were all reduced in plasma from BC patients, compared with healthy controls. Interestingly, these ratios of methylated adenosine nucleosides to adenosine in plasma could better discriminate BC patients from healthy controls, compared to the levels of these nucleosides. The present study not only suggests these modified adenosines can act as noninvasive biomarkers of BC but also will contribute to investigating the impacts of RNA methylation on the occurrence and development of BC.

Quantitative analysis of 20 purine and pyrimidine metabolites by HILIC-MS/MS in the serum and hippocampus of depressed mice

Purine and pyrimidine metabolism are vital metabolic pathways in the development, proliferation or repairment of cells or tissues associated with various diseases. Here, a simple, all-in-one injection hydrophilic interaction liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of 20 metabolites: adenine, adenosine, deoxyadenosine, adenosine 5′-monophosphate, cyclic adenosine monophosphate, hypoxanthine, xanthine, inosine, deoxyinosine, xanthosine, xanthosine 5′-monophosphate and uric acid, which are products of purine metabolism; uridine, deoxyuridine, uridine 5′-monophosphate and uracil, are products of pyrimidine metabolism; and corticosterone, methionine, acetylcholine and serotonin. To minimize interference of endogenous molecules in sample matrixes, a combination of activated carbon adsorption and a serum substitute matrix (5% bovine serum albumin in phosphate buffered saline) was utilized and jointly applied. The sensitivity, linearity, stability, precision, accuracy and extraction recovery were evaluated, and the method was demonstrated to be accurate, sensitive and reliable. An analytical strategy was successfully applied to quantitatively determine 20 metabolite levels in the serum and hippocampus of mice with chronic social defeat stress-induced depression. The results showed greatly perturbed purine metabolism in the depressed mice, which was primarily characterized by dramatic increases in hypoxanthine, xanthine and inosine in serum and reduced levels of adenine, adenosine and adenosine 5′-monophosphate in the hippocampus. These findings suggest that this novel strategy can facilitate the quantitative analysis of adenine and other purine and pyrimidine metabolites in tissue and serum and exhibits great potential in the exploration of metabolism-related mechanisms of relevant diseases.

A sensitive HILIC-MS/MS method for quantification of theanine in rat plasma and tissues: Application to preclinical pharmacokinetic study

Adolescent depression is a significant public health problem, with the major depressive disorder having been the leading risk factor for suicide and death amongst children and adolescents. For treating depression, antidepressants are used with minimal clinical evidence data and uncertain efficacy in children. L-theanine has anti-depression and other physiological functions. However, few reports are available on the pharmacokinetics of L-theanine, especially in children and adolescents. In this study, a rapid and sensitive hydrophilic interaction liquid chromatography-tandem mass spectrometry method was established and validated for the quantification of L-theanine in juvenile rat plasma and tissues. Chromatographic separation was conducted via an Agilent ZORBAX HILIC plus column in gradient elution mode. L-theanine and [2H5]-L-glutamic acid (internal standard) were determined under the multi-reaction monitoring mode transitions of m/z 175.0 → 157.9 and m/z 153.0 → 88.2 in positive ionisation mode, respectively, and completed methodology verification. In addition, 10 and 35 mg kg−1 of L-theanine were given by intragastric administration to determine the brain and plasma pharmacokinetic characteristics in healthy and chronic unpredictable mild stress rats, respectively. The distribution of tissues and the limbic system were measured at the same time. The results showed that juvenile and diseased rats have higher absorption than adult rats, and age, dosage and health status could affect the process of L-theanine in vivo. L-theanine also has a high degree of tissue distribution. This study lays a foundation for the clinical treatment of depression in children and adolescents.

Simultaneous solvent extraction and quantification of eleven amine compounds related to Chemical Weapon Convention in soils via hydrophilic interaction liquid chromatography-tandem mass spectrometry

The detection of Chemical Weapon Convention (CWC)-related amine compounds including the precursors or degradation products of V-type organophosphorus nerve agent, nitrogen mustard and 3-quinuclidinyl benzilate is an important aspect for verifying their intact chemical warfare agents. This work focuses on the development of a novel formulation for the simultaneous solvent extraction of eleven CWC-related amine compounds, from the four-type soil matrices including environmental standard soil, sand, clay, and loam. Extracts were well separated on the hydrophilic interaction liquid chromatography (HILIC) and then detected by MS/MS multiple reaction monitoring mode. The type and component of solvent mixtures were optimized to cover a wide range of polarity over all eleven amine compounds with high extraction efficiencies. Extraction parameters, such as the proportion of methanol, water and NH4OH, the times and the period of extraction, and volumes of extraction solution were optimized. The results indicated that a mixed solvent of methanol/water (44:53, v/v) in 3.0% NH4OH was the optimal formulation for extraction of all 11 analytes with high mean extraction recoveries (64.4–96.1%). Specificity and sensitivity were well improved by the good separation of 11 analytes from four-type soil matrices using these optimized HILIC parameters. This method was fully validated for each analyte in four soil matrices. The linear range of 11 analytes was 0.50/0.75–500 ng·g−1 with correlation coefficient (R2) ≥0.990, and intra/inter-day accuracies were 70.3–125% with relative standard deviation (RSD) ≤19.3%. Limit of detection (LOD) of 11 analytes ranged from 0.01 to 0.5 ng·g−1, which was far lower than those reported in previous studies. The built method accomplishes simultaneously quantitative and trace measurement of all eleven CWC-related amine compounds within a single solvent extraction and detection. It only takes a small amount of soil samples and possesses the highest sensitivity over all previous methods. This study provides an optional recommended operating procedure for determination of CWC-related amine compounds in four typical types of complex soils during chemical weapons verification.

Hydrophilic interaction LC-MS/MS method to avoid endogenous interference in the analysis of 4-hydroxy isoleucine from dietary supplementation of fenugreek.

4-Hydroxy isoleucine is one of the potent hypoglycemic active constituents of fenugreek seeds. A method capable of reducing biological interferences is required for bioavailability studies. An isocratic separation of 4-hydroxy isoleucine from endogenous interferences was achieved in ZIC-cHILIC column using 0.1% formic acid in water and acetonitrile (20:80, % v/v) pumped at 0.5ml/min. Quantification was performed in multiple reaction monitoring mode using the transitions of m/z 148.1→102.1 and m/z 276.1→142.2 for 4-hydroxy isoleucine and homatropine (as internal standard), respectively. After full method validation, 4-hydroxy isoleucine levels in human plasma and commercial fenugreek formulations were determined. This method showed good linearity in the range of 50-2000ng/mL. Intra- and interday accuracies were in the range of 90.64-109.0% and precision was<4.82% CV. The mean (SD) plasma concentration of 4-hydroxy isoleucine in healthy individuals at 2h after oral administration of fenugreek tablet was found to be 1590 (260) ng/mL. Half of marketed formulations were found to contain<0.05% of 4-hydroxy isoleucine content. We developed a rapid hydrophilic interaction liquid chromatography-tandem mass spectrometry method for analysis of 4-hydroxy isoleucine in human plasma. This method can be applied directly to conduct the clinical pharmacokinetics studies of 4-hydroxy isoleucine in human population.

Magnetic Amino-Modified Multiwalled Carbon Nanotube (MWCNT) Based Magnetic Dispersive Solid-Phase Extraction (m-dSPE) for the Determination of Paralytic Shellfish Toxins in Bivalve Mollusks with Hydrophilic Interaction Liquid Chromatography–Tandem Mass Spectrometry (HILIC-MS/MS)

A sensitive and efficient magnetic amino modified multi-walled carbon nanotubes (m-MWCNT-NH2)-based magnetic dispersive solid-phase extraction (m-dSPE) coupled with hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) strategy is reported for the simultaneous determination of 13 paralytic shellfish toxins (PSTs) in bivalve mollusks. The samples were extracted twice with 1% acetic acid and purified using optimized m-dSPE procedures with Fe3O4 coated MWCNT-NH2 composites as magnetic adsorbents. The adsorbed PSTs on the m-MWCNT-NH2 adsorbents were separated using a Ni-coated neodymium magnet and subsequently eluted with 2 mL water–acetonitrile–acetic acid (80:20:1, v/v/v) and analyzed by HILIC-MS/MS. The correlation coefficients (r) of the targeted toxins obtained in matrix-matched external standard curves were from 0.997 to 0.999. The PSTs were acquired and quantified in the multiple reaction-monitoring mode and the limits of detection and quantitation in bivalve mollusks were 1.10 to 4.51 μg/kg and 3.67 to 13.5 μg/kg, respectively. Spiked recoveries at three concentrations in negative samples were from 73.4% to 92.5% with precision less than 10.6%. The method validation was in accordance with 2002/657/EC guidelines and method application in commercial bivalve mollusk samples. The developed method was rapid, convenient and economical for the determination of the targeted toxins.

Quantitative Analysis of Methylated Adenosine Modifications Revealed Increased Levels of N 6-Methyladenosine (m6A) and N 6,2'-O-Dimethyladenosine (m6Am) in Serum From Colorectal Cancer and Gastric Cancer Patients.

Colorectal cancer and gastric cancer are the most prevalent gastrointestinal malignancies worldwide, and early detection of these cancers is crucial to reduce their incidence and mortality. RNA methylation plays an important regulatory role in a variety of physiological activities, and it has drawn great attention in recent years. Methylated adenosine (A) modifications such as N6-methyladenosine (m6A), N1-methyladenosine (m1A), 2′-O-methyladenosine (Am), N6,2′-O-dimethyladenosine (m6Am), and N6,N6-dimethyladenosine (m62A) are typical epigenetic markers of RNA, and they are closely correlated to various diseases including cancer. Serum is a valuable source of biofluid for biomarker discovery, and determination of these adenosine modifications in human serum is desirable since they are emerging biomarkers for detection of diseases. In this work, a targeted quantitative analysis method using hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC-MS/MS) was developed and utilized to analyze these methylated adenosine modifications in serum samples. The concentration differences between the healthy volunteers and cancer patients were evaluated by Mann–Whitney test, and receiver operator characteristic (ROC) curve analysis was performed to access the potential of these nucleosides as biomarkers. We demonstrated the presence of the m6Am in human serum for the first time, and we successfully quantified the concentrations of A, m6A, m1A, and m6Am in serum samples from 99 healthy controls, 51 colorectal cancer patients, and 27 gastric cancer patients. We found that the levels of m6A and m6Am in serum were both increased in colorectal cancer or gastric cancer patients, compared to that in healthy controls. These results indicate that m6A and m6Am in serum may act as potential biomarkers for early detection and prognosis of colorectal cancer and gastric cancer. In addition, the present work will stimulate investigations on the effects of adenosine methylation on the initiation and progression of colorectal cancer and gastric cancer.

Comparison between mouse bioassay and HILIC-MS/MS for quantification of paralytic shellfish toxin in Japanese basket clams and mussels caught off coastal Osaka Bay in Japan

ABSTRACT The content and composition of paralytic shellfish toxins (PSTs) in Japanese basket clam (Corbicula japonica) and mussels (Mytilus galloprovincialis) from Osaka Bay, Japan, were investigated using a mouse bioassay (MBA) and hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS), and the association between toxicity values of MBA and HILIC-MS/MS was verified based on research data. The overall toxicity in Japanese basket clam was lower than that in the mussel. The PSTs of Japanese basket clam and mussel consisted mainly of C1, C2, and gonyautoxins 1–4 (GTX1–4) taking toxins compositional differences as mol%. When multiplying the content of different toxins by the toxic equivalent factor (TEF), C2 and GTX1–4 accounted for more than 90% of total toxicity (MU TEF/g) based on the MU TEF score converted by TEF for the two species. The total content of C2 and GTX1–4 converted to toxicity was significantly correlated with the toxicity determined by MBA for the two species (r 2 > 0.983). This study provides a suitable and ethical monitoring method to investigate toxicity in bivalves contaminated with A. tamarense by analysis of only predominant toxins, along with reducing use of MBA.

Determination of Paralytic Shellfish Toxins in Bivalve Mollusks by Amino-Modified Multiwalled Carbon Nanotube (MWCNT) Solid-Phase Extraction (SPE) and High-Performance Liquid Chromatography–Tandem Mass Spectrometry (HPLC–MS/MS)

An amino-modified multiwalled carbon nanotube (MWCNT) solid-phase extraction-based hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for simultaneous determination of 13 paralytic shellfish toxins (PSTs) in bivalve mollusks. Shellfish samples were extracted with 1% acetic acid followed by cleanup using optimized MWCNT-NH2 based solid-phase extraction. Absorbed PSTs were eluted with 2 mL 1% acetic acid–acetonitrile (70/30, v/v). The separation was performed on a Waters BEH Amide column within 7.5 min utilizing 0.1% formic acid–acetonitrile and 2 mM ammonium acetate containing 0.1% formic acid as the mobile phase. The analytes were quantified by matrix-matched external standard curves with correlation coefficients® exceeding 0.997 acquired in multiple reaction-monitoring mode. The limits of detection and quantitation in shellfish samples were from 2.76 to 9.8 μg/kg and 9.11 to 39.3 μg/kg, respectively. Recoveries at three spiked levels ranged from 71.4% to 97.8% with relative standard deviations less than 12.6%. The developed method was satisfactorily applied to the analysis of shellfish.

Kamikihito, a traditional Japanese Kampo medicine, increases the secretion of oxytocin in rats with acute stress

Ethnopharmacological relevanceKamikihito (KKT) is a Kampo medicine that is prescribed in Japan for the treatment of anemia, insomnia and mental anxiety in Japan. However, its precise mechanism of action remains unclear. Aim of the studyThis study aimed to evaluate the possible antistress effect of KKT in rats with acute stress and the contribution of oxytocin to the process. Materials and methodsAcute immobilization stress (AIS; for 90 min) was used to assess the effect of KKT on acute stress. Male Wistar rats were orally treated with KKT. Parameters of stress were evaluated, and concentrations of oxytocin in plasma and cerebrospinal fluid (CSF) were measured. ResultsAIS-induced defecation and fecal weight were significantly decreased because of treatment with KKT. The plasma levels of stress-related hormones following AIS were investigated. The pre-administration of KKT significantly increased adrenocorticotrophic hormone (ACTH) and corticosterone (CORT) levels following AIS. Conversely, there was no significant change in the plasma oxytocin level. Microdialysis and hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) were used to monitor the oxytocin secretion in CSF. Oxytocin level increased during AIS following the treatment of KKT. At 30 min after AIS, the level remained higher than before AIS. Furthermore, using an open field test, the locomotion (exploratory behavior) immediately after AIS was examined. The total traveled distance decreased after AIS; however, the decrease was significantly inhibited by the treatment of KKT. However, the effect of KKT was obstructed by the pre-administration of the oxytocin receptor antagonist. ConclusionsThese results suggest that KKT has antistress activity and increased oxytocin secretion may be a mechanism underlying this phenomenon.

Effects of Chronic Secondhand Smoke (SHS) Exposure on Cognitive Performance and Metabolic Pathways in the Hippocampus of Wild-Type and Human Tau Mice.

Background:Exposure to secondhand smoke (SHS) is a risk factor for developing sporadic forms of sporadic dementia. A human tau (htau) mouse model is available that exhibits age-dependent tau dysregulation, neurofibrillary tangles, neuronal loss, neuroinflammation, and oxidative stress starting at an early age (3–4 months) and in which tau dysregulation and neuronal loss correlate with synaptic dysfunction and cognitive decline.Objective:The goal of this study was to assess the effects of chronic SHS exposure (10 months’ exposure to ) on behavioral and cognitive function, metabolism, and neuropathology in mice.Methods:Wild-type (WT) and htau female and male mice were exposed to SHS (90% side stream, 10% main stream) using the SCIREQ® inExpose™ system or air control for 168 min per day, for 312 d, 7 d per week. The exposures continued during the days of behavioral and cognitive testing. In addition to behavioral and cognitive performance and neuropathology, the lungs of mice were examined for pathology and alterations in gene expression.Results:Mice exposed to chronic SHS exposure showed the following genotype-dependent responses: a) lower body weights in WT, but not htau, mice; b) less spontaneous alternation in WT, but not htau, mice in the Y maze; c) faster swim speeds of WT, but not htau, mice in the water maze; d) lower activity levels of WT and htau mice in the open field; e) lower expression of brain PHF1, TTCM1, , and HSP90 protein levels in WT male, but not female, mice; and f) more profound effects on hippocampal metabolic pathways in WT male than female mice and more profound effects in WT than htau mice.Discussion:The brain of WT mice, in particular WT male mice, might be especially susceptible to the effects of chronic SHS exposure. In WT males, independent pathways involving ascorbate, flavin adenine dinucleotide, or palmitoleic acid might contribute to the hippocampal injury following chronic SHS exposure. https://doi.org/10.1289/EHP8428

Analytical characterization of DNA and RNA oligonucleotides by hydrophilic interaction liquid chromatography-tandem mass spectrometry

Liquid chromatography-mass spectrometry has been widely implemented as a powerful tool for providing in-depth characterization of nucleic acid therapeutic modalities, such as anti-sense oligonucleotides and small interfering RNAs (siRNAs). In this study, we developed a generic hydrophilic interaction liquid chromatography (HILIC) hyphenated with tandem mass spectrometry method in the absence of ion-pairing reagents and demonstrated its capability as an attractive and robust alternative for oligonucleotide and siRNA analysis. HILIC separation of mixtures of unmodified and fully phosphorothioate-modified DNA oligonucleotides and their synthetic 3’ exonuclease-digested metabolites were also assessed. High-resolution mass spectrometric (HRMS) analysis was used to determine the deconvoluted masses of oligonucleotide and siRNA standards and their impurities. To enable unbiased sequence characterization with tandem mass spectrometry (MS/MS), we also optimized higher-energy C-trap dissociation (HCD) on improving the sequence coverage of DNA and RNA oligonucleotides. Lastly, we evaluated on-column sensitivity for a phosphorothioate oligonucleotide by performing targeted analysis with either targeted selected ion monitoring (tSIM) or parallel reaction monitoring (PRM). Higher on-column sensitivity of 13 ng, equivalent to 2.0 pmol, of a phosphorothioate oligonucleotide was achieved by tSIM analysis as compared to PRM analysis.

An efficient HILIC-MS/MS method for the trace level determination of three potential genotoxic impurities in aripiprazole active drug substance

A sensitive and selective hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) method was developed and validated for trace analysis of potential genotoxic impurities (PGIs): 2,3-dichloroaniline (PGI-1), bis(2-chloroethyl) amine (PGI-2), and 2-chloroethylamine (PGI-3), in aripiprazole (APZ) active drug substance. Separation of analytes was achieved on ACE HILIC–N Column (HILN-5-1046U, 100 × 4.6 mm, 5 μm) in gradient elution mode with mobile phase A [acetonitrile:ammonium formate buffer (95:5 v/v)] and mobile phase B [acetonitrile:ammonium formate buffer (50:50 v/v)] at a flow rate of 0.8 mL/min. Developed method was linear in the concentration range of 8–100 ppm for PGI-1, 11–100 ppm for PGI-2, and 12.5–100ppm for PGI-3 with R2 > 0.996. The developed method was accurate for quantification of each PGI with percent recoveries greater than 96% and RSD (%) not more than 5%. The developed method was precise for quantification of PGIs in aripiprazole with RSD (%) of not more than 4% for any of the PGIs. There was no interference of diluent peaks at the retention time of the PGIs and APZ in the method. All the PGIs and sample solutions were found to be stable at ambient laboratory temperature (25 ± 5 °C) and refrigerated condition (2–8 °C) for a period of 48 h. The developed HILIC-MS/MS method can be used for trace quantification of PGIs in aripiprazole drug in quality control laboratories of the pharmaceutical industry.

Plasma Phospholipidomic Profile Differs between Children with Phenylketonuria and Healthy Children.

Phenylketonuria (PKU) is a disease of the catabolism of phenylalanine (Phe), caused by an impaired function of the enzyme phenylalanine hydroxylase. Therapeutics is based on the restriction of Phe intake, which mostly requires a modification of the diet. Dietary restrictions can lead to imbalances in specific nutrients, including lipids. In the present study, the plasma phospholipidome of PKU and healthy children (CT) was analyzed by hydrophilic interaction liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. Using this approach, 187 lipid species belonging to nine different phospholipid classes and three ceramides were identified. Principal component analysis of the lipid species data set showed a distinction between PKU and CT groups. Univariate analysis revealed that 146 species of phospholipids were significantly different between both groups. Lipid species showing significant variation included phosphatidylcholines, containing polyunsaturated fatty acids (PUFA), which were more abundant in PKU. The high level of PUFA-containing lipid species in children with PKU may be related to a diet supplemented with PUFA. This study was the first report comparing the plasma polar lipidome of PKU and healthy children, highlighting that the phospholipidome of PKU children is significantly altered compared to CT. However, further studies with larger cohorts are needed to clarify whether these changes are specific to phenylketonuric children.

In vitro and in vivo analysis of metabolites involved in the TCA cycle and glutamine metabolism associated with cisplatin resistance in human lung cancer

ABSTRACT Elucidating the dysregulated metabolic pathways in cancer cells and their relevance to cisplatin resistance could yield new insights into cancer therapy. We previously reported that eight metabolites involved in the tricarboxylic acid (TCA) cycle and glutamine metabolism were associated with platinum-based chemotherapy efficacy in human lung cancer. Here, we investigated the metabolic differences upon cisplatin treatment in lung cancer in vitro and in vivo. A simple and partially validated standard addition method was applied for the quantification of five metabolites involved in the TCA cycle and glutamine metabolism using amide hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The present study investigated the levels of these biomarkers in A549 cells and the cisplatin-resistant A549-DDP cells, as well as in the plasma before and after cisplatin treatment in A549 xenograft mice. Levels of five metabolites, including 2-hydroxyglutaric acid (2-HG), α-ketoglutarate (α-KG), succinate, glutamine, and glutamate, showed a decreasing trend in A549-DDP cells. In addition, 2-HG and glutamine were the most significantly altered metabolites in cisplatin-treated A549 xenograft mice. These data indicate that the TCA cycle and glutamine metabolism play important roles in cisplatin-based chemotherapy resistance in lung cancer. Our results provide a new angle for exploring the molecular mechanisms underlying cisplatin resistance.

A new high-throughput method based on hydrophilic interaction liquid chromatography-tandem mass spectrometry to determine 18 short-chain carboxylic acids in foods

In this work, an interesting analytical method to determine 18 short-chain carboxylic acids in food samples was developed using Hydrophilic Interaction Liquid Chromatography coupled to mass spectrometry (HILIC-MS/MS). The method was optimized through univariate and multivariate designs. The chromatographic SeQuant® ZIC®-HILIC column and mobile phase comprised of 75:25 (v/v) ACN and ammonium formate buffer (8 mmol L−1, pH 3) were selected. The analytical performance was assessed obtaining limits of quantification that varied from 0.04 to 2 mg L−1, and precision values less than 14%. Five different food samples were analyzed using calibration by standard addition with internal standard and sample preparation by dilute-and-shoot, which allowed for an efficient determination of the analytes in matrices of high complexity. In summary, the proposed method provided high-throughput, a great selectivity given by the HILIC-MS/MS system, an adequate sensitivity for the tested samples and interesting information about HILIC mechanisms for short-chain carboxylic acids as analytes and zwitterionic stationary phase.

A novel derivatization strategy for profiling phosphate ester/anhydride metabolic network and application on glioma rats using HILIC-MS/MS

Phosphate esters and anhydrides have great significance in the field of biochemical research and medical therapy. The genetic materials (DNA or RNA), most of the coenzymes, many intermediary metabolites, such as nucleotides and glycosyl phosphates in vivo are phosphodiesters, phosphoric acid or phosphates, respectively. It is important to monitor endogenous active phosphate metabolites for investigating many biological processes or drug mechanism. However, the detection and determination of those free active phosphate metabolites are challenged due to their unstable and easily hydrolyzed property and relatively low sensitivity, especially diphosphates and triphosphates. In the current study, we successfully developed a strategy by 3-aminomethyl pyridine (AMPy) derivatization coupled with hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS) for simultaneous determination of multiple types of phosphate metabolites with good stability in 48 h and 29 to 126-fold improvement of the limit of detection (LOD). Based on the diagnostic fragment ions of different types of AMPy-derivatized phosphate metabolites, characteristic MRM ion pairs were successfully performed for global profiling of the phosphate metabolites in phosphate ester/anhydride metabolic network, including nucleotide/deoxynucleotide mono/di/triphosphates, glycosyl mono/diphosphates, and other key phosphates, such as 5-phosphoribosyl-1-pyrophosphate (PRPP), SAICARP and FAICARP in HPF, HUVEC and PBMCs cells without standards. The developed strategy greatly expanded the coverage of applying a single derivatization reaction to analyze active phosphate metabolites. Finally, the established method was performed to investigate the phosphate esters and anhydrides based on a glioma rat model. For the first time, phosphate metabolites were comprehensively characterized based on phosphate ester and anhydride metabolic network, covering nucleotide metabolism, glycolysis and pentose phosphate pathways, etc. The results demonstrated that the applicability of the method could be extended to a wider range of active phosphate compounds and could facilitate to related applications in the future studies.

Automated On-Line Isolation and Fractionation System for Nanosized Biomacromolecules from Human Plasma.

An automated on-line isolation and fractionation system including controlling software was developed for selected nanosized biomacromolecules from human plasma by on-line coupled immunoaffinity chromatography-asymmetric flow field-flow fractionation (IAC-AsFlFFF). The on-line system was versatile, only different monoclonal antibodies, anti-apolipoprotein B-100, anti-CD9, or anti-CD61, were immobilized on monolithic disk columns for isolation of lipoproteins and extracellular vesicles (EVs). The platelet-derived CD61-positive EVs and CD9-positive EVs, isolated by IAC, were further fractionated by AsFlFFF to their size-based subpopulations (e.g., exomeres and exosomes) for further analysis. Field-emission scanning electron microscopy elucidated the morphology of the subpopulations, and 20 free amino acids and glucose in EV subpopulations were identified and quantified in the ng/mL range using hydrophilic interaction liquid chromatography-tandem mass spectrometry (HILIC-MS/MS). The study revealed that there were significant differences between EV origin and size-based subpopulations. The on-line coupled IAC-AsFlFFF system was successfully programmed for reliable execution of 10 sequential isolation and fractionation cycles (37–80 min per cycle) with minimal operator involvement, minimal sample losses, and contamination. The relative standard deviations (RSD) between the cycles for human plasma samples were 0.84–6.6%.

A Hydrophilic Interaction Liquid Chromatography-Tandem Mass Spectrometry Quantitative Method for Determination of Baricitinib in Plasma, and Its Application in a Pharmacokinetic Study in Rats.

Baricitinib, is a selective and reversible Janus kinase inhibitor, is commonly used to treat adult patients with moderately to severely active rheumatoid arthritis (RA). A fast, reproducible and sensitive method of liquid chromatography-tandem mass spectrometry (LC-MS/MS) for the quantification of baricitinib in rat plasma has been developed. Irbersartan was used as the internal standard (IS). Baracitinib and IS were extracted from plasma by liquid–liquid extraction using a mixture of n-hexane and dichloromethane (1:1) as extracting agent. Chromatographic separation was performed using Acquity UPLC HILIC BEH 1.7 µm 2.1 × 50 mm column with the mobile phase consisting of 0.1% formic acid in acetonitrile and 20 mM ammonium acetate (pH 3) (97:3). The electrospray ionization in the positive-mode was used for sample ionization in the multiple reaction monitoring mode. Baricitinib and the IS were quantified using precursor-to-production transitions of m/z 372.15 > 251.24 and 429.69 > 207.35 for baricitinib and IS, respectively. The method was validated according to the recent FDA and EMA guidelines for bioanalytical method validation. The lower limit of quantification was 0.2 ng/mL, whereas the intra-day and inter-day accuracies of quality control (QCs) samples were ranged between 85.31% to 89.97% and 87.50% to 88.33%, respectively. Linearity, recovery, precision, and stability parameters were found to be within the acceptable range. The method was applied successfully applied in pilot pharmacokinetic studies.

Halomethanesulfonic Acids-A New Class of Polar Disinfection Byproducts: Standard Synthesis, Occurrence, and Indirect Assessment of Mitigation Options.

Halomethanesulfonic acids (HMSAs) are recently discovered polar disinfection byproducts without commercially available reference materials. To allow for their accurate quantification, we successfully synthesized standards for the four presumably most prevalent HMSA congeners: chloromethanesulfonic acid, bromomethanesulfonic acid, dichloromethanesulfonic acid, and bromochloromethanesulfonic acid. After structure confirmation and quantification with high-resolution mass spectrometry and nuclear magnetic resonance spectroscopy, we integrated them into a multilayer solid phase extraction and hydrophilic interaction liquid chromatography-tandem mass spectrometry method dedicated to the analysis of polar water contaminants. With this method we monitored HMSAs in drinking water production plants from four European countries and tap water samples taken in six countries. HMSAs were detected in the low μg/L range after the chlorination step during drinking water production, all tap waters samples, and two surface waters used for drinking water production. Concentrations in tap water samples ranged from 0.07 μg/L to 11.5 μg/L while the HMSA concentrations in surface waters were in the range of 100 ng/L. We utilized the HMSA formation potential to indirectly assess the behavior of hitherto unknown HMSA precursors, consequently identifying ozonation, filtration through activated carbon, and reverse osmosis as efficient removal tools for HMSA precursors, thus limiting their formation during subsequent water disinfection.