Hydrophilic Interaction Ultra-high Performance Liquid Research Articles

Cost-Effective Simultaneous Determination of τ- and π-Methylhistidine in Dairy Bovine Plasma from Large Cohort Studies Using Hydrophilic Interaction Ultra-High Performance Liquid Chromatography Coupled to Tandem Mass Spectrometry

The isomeric metabolites τ- and π-methylhistidine (formerly referred to as 3- and 1-methylhistidine) are known biomarkers for muscle protein breakdown and meat protein intake, frequently used in studies involving humans and animals. In the present study, we report the development and validation of a simple HILIC-MS/MS method for individual determination of τ-MH and π-MH in a large cohort of blood plasma samples from dairy cows. Their separate determination was achieved mainly through a mass spectrometry fragment ion study, which revealed that the two isomers exhibited distinct mass spectrometric behaviors at different collision energies. Chromatographic conditions were optimised to achieve better separation, minimizing inter-channel interference to less than 1% in both directions. A simple and effective sample clean-up method facilitated low laboratory manual workload. The analytical method was validated for the determination of τ-MH and π-MH in bovine plasma within a concentration range of 80 to 1600 μg/L and provided good linearity (>0.99 for both curves) and precision (<10%). Overall, the developed method enabled the determination of the two isomers in an efficient and economic-friendly manner suitable for large cohort bovine studies (involving hundreds to thousands of samples) mainly to provide data for statistical use.

High-throughput immunoaffinity enrichment and N-glycan analysis of human plasma haptoglobin.

Haptoglobin (Hp) is a positive acute phase protein, synthesized in the liver, with four N-glycosylation sites carrying mainly complex type N-glycans. Its glycosylation is altered in different types of diseases but still has not been extensively studied mainly due to analytical challenges, especially the lack of a fast, efficient, and robust high-throughput Hp isolation procedure. Here, we describe the development of a high-throughput method for Hp enrichment from human plasma, based on monolithic chromatographic support in immunoaffinity mode and downstream Hp N-glycome analysis by hydrophilic interaction ultrahigh-performance liquid chromatography with fluorescent detection (HILIC-UHPLC-FLR). Chromatographic monolithic supports in a 96-well format enable fast, efficient, and robust Hp enrichment directly from diluted plasma samples. The N-glycome analysis demonstrated that a degree of Hp deglycosylation differs depending on the conditions used for N-glycan release and on the specific glycosylation site, with Asn 241 being the most resistant to deglycosylation under tested conditions. HILIC-UHPLC-FLR analysis enables robust quantification of 28 individual chromatographic peaks, in which N-glycan compositions were determined by UHPLC coupled to electrospray ionization quadrupole time of flight mass spectrometry. The developed analytical approach enables fast evaluation of total Hp N-glycosylation and is applicable in large-scale studies.

Free amino acids in African indigenous vegetables: Analysis with improved hydrophilic interaction ultra-high performance liquid chromatography tandem mass spectrometry and interactive machine learning

A hydrophilic interaction (HILIC) ultra-high performance liquid chromatography (UHPLC) with triple quadrupole tandem mass spectrometry (MS/MS) method was developed and validated for the quantification of 21 free amino acids (AAs). Compared to published reports, our method renders collectively improved sensitivity with lower limit of quantification (LLOQ) at 0.5~42.19 ng/mL with 0.3 μL injection volume (or equivalently 0.15~12.6 pg injected on column), robust linear range from LLOQ up to 3521~5720 ng/mL (or 1056 ~ 1716 pg on column) and a high throughput with total time of 6 min per sample, as well as easier experimental setup, less maintenance and higher adaptation flexibility. Ammonium formate in the mobile phase, though commonly used in HILIC, was found unnecessary in our experimental setup, and its removal from mobile phase was key for significant improvement in sensitivity (4~74 times higher than with 5 mM ammonium formate). Addition of 10 (or up to100 mM) hydrochloric acid (HCl) in the sample diluent was crucial to keep response linearity for basic amino acids of histidine, lysine and arginine. Different HCl concentration (10~100 mM) in sample diluent also excreted an effect on detection sensitivity, and it is of importance to keep the final prepared sample and calibrators in the same HCl level. Leucine and isoleucine were distinguished using different transitions. Validated at seven concentration levels, accuracy was bound within 75~125%, matrix effect generally within 90~110%, and precision error mostly below 2.5%. Using this newly developed method, the free amino acids were then quantified in a total of 544 African indigenous vegetables (AIVs) samples from African nightshades (AN), Ethiopian mustards (EM), amaranths (AM) and spider plants (SP), comprising a total of 8 identified species and 43 accessions, cultivated and harvested in USA, Kenya and Tanzania over several years, 2013~2018. The AN, EM, AM and SP were distinguished based on free AAs profile using machine learning methods (ML) including principle component analysis, discriminant analysis, naïve Bayes, elastic net-regularized logistic regression, random forest and support vector machine, with prediction accuracy achieved at ca. 83~97% on the test set (train/test ratio at 7/3). An interactive ML platform was constructed using R Shiny at https://boyuan.shinyapps.io/AIV_Classifier/ for modeling train-test simulation and category prediction of unknown AIV sample(s). This new method presents a robust and rapid approach to quantifying free amino acids in plants for use in evaluating plants, biofortification, botanical authentication, safety, adulteration and with applications to nutrition, health and food product development.

Core-Fucosylated Tetra-Antennary N-Glycan Containing A Single N-Acetyllactosamine Branch Is Associated with Poor Survival Outcome in Breast Cancer.

(1) Glycoproteins account for ~80% of proteins located at the cell surface and in the extracellular matrix. A growing body of evidence indicates that α-L-fucose protein modifications contribute to breast cancer progression and metastatic disease. (2) Using a combination of techniques, including matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI-IMS) based in cell and on tissue imaging and glycan sequencing using exoglycosidase analysis coupled to hydrophilic interaction ultra-high performance liquid chromatography (HILIC UPLC), we establish that a core-fucosylated tetra-antennary glycan containing a single N-acetyllactosamine (F(6)A4G4Lac1) is associated with poor clinical outcomes in breast cancer, including lymph node metastasis, recurrent disease, and reduced survival. (3) This study is the first to identify a single N-glycan, F(6)A4G4Lac1, as having a correlation with poor clinical outcomes in breast cancer.

Non-targeted screening and determination of steviol glycosides in Chinese liquor and beverages by ultra high performance liquid chromatography-quadrupole/Orbitrap mass spectrometry

A method for simultaneous screening and determination of steviol glycosides in Chinese liquor and beverages by hydrophilic interaction ultra-high performance liquid chromatography-quadrupole/Orbitrap high resolution mass spectrometry (HILIC-UHPLC-Q/Orbitrap HRMS) was developed. In addition, the MS fragment path ways of steviol glycosides were characterized. The sample was diluted and filtrated before analysis, separated by a Waters Xbridge Amide column (150 mm×4.6 mm, 3.5 μm), and determined via UHPLC-Q/Orbitrap HRMS. The analytes were electronic using electro spray ion source in the negative mode, and the data were collected using full mass scan and data-dependent MS2 scan. Good linear relationships (10-1000 μg/L) were obtained. The limits of detection ranged from 0.3 to 20 μg/L. The mean recoveries varied from 81.9% to 106% at the spiked levels of 10-100 μg/kg, and the relative standard deviations ranged from 0.1% to 9.3% (n=3). Two characteristic fragment ions of steviol glycosides were identified by the analysis of fragment ion spectra of eight types of steviol glycosides standards. Non-targeted screening of unknown steviol glycosides in samples was carried out on the basis of the identified characteristic fragment ions. The proposed method is comprehensive, accurate, and sensitive for rapid screening and determination of sweeteners in food samples.

Metabonomic Evaluation of Chronic Unpredictable Mild Stress-Induced Changes in Rats by Intervention of Fluoxetine by HILIC-UHPLC/MS

Hydrophilic interaction-ultra high performance liquid chromatography (HILIC-UHPLC) allows the analysis of highly polar metabolites, providing complementary information to reversed-phase (RP) chromatography. By optimization of the preparation and analytical conditions in HILIC mode, HILIC-UHPLC/MS was applied for the global metabolic profiling of rat plasma samples generated in an experimental model of chronic unpredictable mild stress (CUMS), and the concomitant investigation of the protective effect of fluoxetine was also evaluated. Identification of plasma metabolic profiles indicated that significant changes in specific metabolites occurred after fluoxetine exposure, including increased phenylalanine, serine, acetyl-L-carnitine, carnitine and decreased creatine, betaine, proline, tryptophan, tyrosine, C16:0 LPC. Some novel biomarkers from this HILIC-UHPLC/MS approach were betaine, proline, tyrosine creatine and serine compared with the results of RP-UHPLC/MS. The complementary nature of this technique is confirmed and is on agreement with previously published studies.

Hydrophilic Interaction Ultra-High Performance Liquid Chromatography Coupled with Triple-Quadrupole Mass Spectrometry for Determination of Nucleosides and Nucleobases in Animal Horns

Traditional Chinese Medicines (TCMs) derived from animal horns are one of the most important types of Chinese medicine. In the present study, a fast and sensitive analytical method was established for qualitative and quantitative determination of 14 nucleosides and nucleobases in animal horns using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadruple tandem mass spectrometry (HILIC-UPLC–QQQ-MS/MS) in selective reaction monitoring (SRM) mode. The method was optimized and validated, and showed good linearity, precision, repeatability, and accuracy. The method was successfully used to determine contents of the 14 nucleosides and nucleobases in 25 animal horn samples. Hierarchical clustering analysis (HCA) and principal component analysis (PCA) were performed and the 25 samples were thereby divided into two groups, which agreed with taxonomy. The method may enable quick and effective search of substitutes for precious horns.

Hydrophilic interaction ultra-high performance liquid chromatography coupled with triple quadrupole mass spectrometry for determination of nucleotides, nucleosides and nucleobases in Ziziphus plants

In this study, a rapid and sensitive analytical method was developed for the determination of 20 nucleobases, nucleosides and nucleotides in Ziziphus plants at trace levels by using hydrophilic interaction ultra-high performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HILIC–UHPLC-TQ-MS/MS) in multiple-reaction monitoring (MRM) mode. Under the optimized chromatographic conditions, good separation for 20 target compounds were obtained on a UHPLC Amide column with sub-2μm particles within 10min. The overall LODs and LOQs were between 0.11–3.12ngmL−1 and 0.29–12.48ngmL−1 for the 20 analytes, respectively. It is the first report about simultaneous analysis of nucleobases, nucleosides and nucleotides in medicinal plants using HILIC–UHPLC-TQ-MS/MS method, which affords good linearity, precision, repeatability and accuracy. The developed method was successfully applied to Ziziphus plant (Z. jujuba, Z. jujuba var. spinosa and Z. mauritiana) samples. The analysis showed that the fruits and leaves of Ziziphus plants are rich in nucleosides and nucleobases as well as nucleotides, and could be selected as the healthy food resources. Our results in present study suggest that HILIC–UHPLC-TQ-MS/MS method could be employed as a useful tool for quality assessment of the samples from the Ziziphus plants as well as other medicinal plants or food samples using nucleotides, nucleosides and nucleobases as markers.

Quantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometry

A hydrophilic interaction liquid chromatography (HILIC)-MS/MS profiling method was developed for the efficient separation and quantification of small polar molecules, mostly primary metabolites. The method was based on an ultrahigh performance liquid chromatography (UHPLC) separation system coupled with ESI mass spectrometry on a triple quadrupole mass spectrometer, operating in both positive and negative ionisation mode using rapid polarity switching. With the developed method quantitation of 135 compounds belonging in four major classes of polar compounds (sugars, aminoacids, organic acids and amines) was achieved in a single run of 30min. The method was applied to grape extracts from different varieties and provided information on primary metabolite content. Multivariate statistical analysis was applied using the concentrations found, with the aim of investigating the differences in metabolite profiles. Classification of grapes according to their skin colour was carried out using principle component analysis based on the concentration variation of a number of the metabolites studied.

Future Developments for Triple Quadrupole Mass Spectrometers: Not Just Hardware

BioanalysisVol. 3, No. 7 EditorialFuture developments for triple quadrupole mass spectrometers: not just hardwarePatrick BennettPatrick BennettThermo Fisher Scientific, 355 River Oaks Parkway, San Jose, CA 95134, USA. Search for more papers by this authorEmail the corresponding author at patrick.bennett@thermofisher.comPublished Online:31 Mar 2011https://doi.org/10.4155/bio.11.41AboutSectionsView ArticleView Full TextPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareShare onFacebookTwitterLinkedInReddit View articleKeywords: future innovationhigh resolutionmatrix effectssystem specializationtriple quadrupoleFiguresReferencesRelatedDetailsCited ByQuantitative profiling of polar primary metabolites using hydrophilic interaction ultrahigh performance liquid chromatography–tandem mass spectrometryJournal of Chromatography A, Vol. 125910years of MS instrumental developments – Impact on LC–MS/MS in clinical chemistryJournal of Chromatography B, Vol. 883-884 Vol. 3, No. 7 Follow us on social media for the latest updates Metrics Downloaded 503 times History Published online 31 March 2011 Published in print April 2011 Information© Future Science LtdKeywordsfuture innovationhigh resolutionmatrix effectssystem specializationtriple quadrupoleFinancial & competing interests disclosureThe author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.No writing assistance was utilized in the production of this manuscript.PDF download