Utilization of a Dextran Ladder to Standardize PGC-LC-MS-Based Glycomics

Porous graphitised carbon (PGC) based chromatographic separation of glycans achieves high-resolution separation of glycan mixtures released from glycoproteins, including structurally similar isomers. While there is some understanding of glycan separation on PGC, system-independent retention values have not been established. Using hydrolysed dextran as an internal standard, and Skyline software for post-acquisition normalisation, retention time and glycan peak area variation of replicate injections of glycan mixtures was significantly reduced. Normalisation of retention time to the dextran ladder allowed assignment of system-independent retention values, values that are applicable to all PGC-based separations regardless of chromatographic system. We have built a library of over 300 PGC-separated glycan structures with assigned normalised glucose unit (GU) PGC retention values. To further define the mechanism of glycan separation with PGC, we identified predictive models for the chromatographic effects resulting from addition and/or removal of core-fucosylation and bisecting GlcNAc based on the PGC normalised retention time library. A dextran ladder spectral library was also built to ensure correct retention time assignment of the internal standard added to glycan mixtures. Using the spectral matching feature in Skyline, isomeric discrimination between O-mannosylated glycans and the glucose-based dextran ladder was achieved. As a result, system-independent automated assignment of glycan structure based on precursor mass and glucose unit value, using a glycan structure reference library, can be achieved using PGC-LC-MS.

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GlycanGUI: Automated Glycan Annotation and Quantification Using Glucose Unit Index

Frontiers in Chemistry ◽

10.3389/fchem.2021.707382 ◽

2021 ◽

Vol 9 ◽

Author(s):

Rui Zhang ◽

Wenjing Peng ◽

Sakshi Gautam ◽

Yifan Huang ◽

Yehia Mechref ◽

...

Keyword(s):

Experimental Data ◽

Retention Time ◽

Laboratory Data ◽

Internal Standard ◽

Software Tool ◽

Graphic User Interface ◽

Liquid Chromatography Mass Spectrometry ◽

Experimental Conditions ◽

Glucose Unit ◽

Unit Index

The retention time provides critical information for glycan annotation and quantification from the Liquid Chromatography Mass Spectrometry (LC-MS) data. However, the variation of the precise retention time of glycans is highly dependent on the experimental conditions such as the specific separating columns, MS instruments and/or the buffer used. This variation hampers the exploitation of retention time for the glycan annotation from LC-MS data, especially when inter-laboratory data are compared. To incorporate the retention time of glycan across experiments, Glucose Unit Index (GUI) can be computed using the dextrin ladder as internal standard. The retention time of glycans are then calibrated with respect to glucose units derived from dextrin ladders. Despite the successful application of the GUI approach, the manual calibration process is quite tedious and often error prone. In this work, we present a standalone software tool GlycanGUI, with a graphic user interface to automatically carry out the GUI-based glycan annotation/quantification and subsequent data analysis. When tested on experimental data, GlycanGUI reported accurate GUI values compared with manual calibration, and thus is ready to be used for automated glycan annotation and quantification using GUI.

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Bioanalytical method development and validation of simultaneous analysis of telmisartan and hydrochlorthiazide in human plasma using LC-MS/MS

International Journal of Bioassays ◽

10.21746/ijbio.2016.03.003 ◽

2016 ◽

Vol 5 (03) ◽

pp. 4862 ◽

Cited By ~ 1

Author(s):

Mathew George* ◽

Lincy Joseph ◽

Arpit Kumar Jain ◽

Anju V.

Keyword(s):

Human Plasma ◽

Retention Time ◽

High Performance ◽

Method Development ◽

Matrix Effects ◽

Solid Phase ◽

Internal Standard ◽

Assay Method ◽

Buffer System ◽

Internal Standards

A simple, sensitive, rapid and economic high performance thin layer chromatographic method and a mass spectroscopic assay method has been developed for the quantification of telmisartan and hydrochlorthiazide combination in human plasma. The internal standards and analytes were extracted from human plasma by solid-phase extraction with HLB Oasis1cc (30mg) catridges. The scanning and optimization for the samples are done using methanol: water (50:50). The samples were chromatographed using reverse phase chromatography with C-18 column of different manufacturers like Ascentis C18 (150×4. 6, 5µ) using the buffer system Acetonitrile: Buffer (80:20%v/v) which consist of 2±0. 1Mm ammonium format at a flow rate of 0. 7ml/min at a column oven temperature 35±10c. The internal standard used was hydrochlorthiazide13c1, d2 and telmisartand3. The extraction techniques include conditioning, loading, washing and elution, drying followed by reconstitution of the dried samples. The volume injected was 10µl with the retention time of 3-4 min for telmisartan, 1-2 min for hydrochlorthiazide and for the internal standards the retention time was 3-4 min for telmisartand3 and 1-2 min for hydrochlorthiazide c13d2. The rinsing solution was Acetonitrile: HPLC grade water in the ratio (50:50). The above developed method was validated using various parameters like selectivity and sensitivity, accuracy and precision, matrix effects, % recovery and various stability studies. The method was proved to be sensitive, accurate, precise and reproducible. The preparation showed high recovery for the quantitative determination of telmisartan and hydrochlorthiazide in human plasma.

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Determination of total proteinogenic amino acids and taurine by pre-column derivatisation and UHPLC: Single Laboratory Validation, First Action 2019.09

Journal of AOAC International ◽

10.1093/jaoacint/qsaa124 ◽

2020 ◽

Author(s):

George Joseph ◽

Asha Varughese ◽

Ann Daniel

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Infant Formula ◽

Retention Time ◽

Sulfonic Acid ◽

Internal Standard ◽

The Other ◽

Method Performance ◽

Single Laboratory

Abstract Background A method has been developed and validated for selective, accurate and precise determination of total proteinogenic amino acids and taurine from Infant Formula and Adult/Pediatric Nutritional Formulas (powders, ready-to-feed liquids, and liquid concentrates). The method was reviewed by the AOAC INTERNATIONAL SPIFAN Expert Review Panel (ERP) during the 133rd AOAC Annual Meeting & Expo on September 7, 2019 in Denver, CO, USA and was recommended to First Action Official MethodsSM status. Objective The method involves protein hydrolysis to amino acids, a simple pre-column derivatisation of amino acids and separation of derivatised amino acids by UHPLC. The quantification of amino acids is performed by multi-point calibration using norvaline as the internal standard. The analytical method is capable of quantitative determination for 22 proteinogenic amino acids, but cannot be used to quantitate tryptophan, which is destroyed during the acid hydrolysis step. Asparagine is determined as aspartic acid and glutamine as glutamic acid. The cystine and cysteine are converted to S-2-carboxyethylthiocysteine (CYSx) and the derivative is separated from the other amino acids. Citrulline which is present in some matrices and it is separated from other amino acids is not included in the method performance evaluation in the single laboratory validation (SLV). Method The proposed method met all the performance requirement limits set in standard method performance requirements (SMPR) 2014.013 for total proteinogenic amino acids and taurine. The correlation coefficient of multi-point calibration was not less than 0.999 for any amino acids at any point in the SLV study confirming the validity of linear dynanic range (LDR) and linearity of the method. The individual amino acids in the chromatogram were identified by absolute retention time and relative retention time (RRT) with respect to the internal standard norvaline. There were no significant (S/N Ratio <10) interferences from the reagents or by-products of derivatisation and targeted matrices. The method demonstrated high selectivity. Result Accuracy of the method was validated using standard reference materials (NIST SRM 1869 and 1849a) and spike recovery studies. The amino acid results in the SRMs were within the ranges of Reference Mass Fraction Values. The accuracy of the method was corroboratively validated by spike recovery studies. The average spike recovery range between 93 to 107% ensure the accuracy of the method for amino acids and compliance to the AOAC SMPR 2014.013. Conclusions Precision data of the method demonstrate that it meets the stakeholder requirements as per the SMPR. The mean RSDr for all the amino acids for 17 matrices selected for the SLV were not more than 4%. The method is very sensitive and the LOQ can go down to approximately ten times lower than the SMPR requirements. The sensitivity of method is a direct reflection of its signal to noise ratio which ensures guaranteed method performance at the lower levels of amino acids in these matrices. Highlights Taurine (aminoethane sulfonic acid) unlike the other amino acids is a beta-sulfonic amino acid that is not used in protein synthesis but is found as a free amino acid in tissues. The acidic functional group (-COOH) in common amino acid is replaced with a sulfonic acid (-SO3H) group in Taurine. The method offers baseline separation of citrulline which is an alpha amino acid generally present in Infant Formula and Adult/Pediatric Nutritional products. The separation of citrulline eliminates the risk of interference of this compound with other amino acids. The method can also separate and quantitate hydroxyproline, an important component of collagen that is often used to quantitate collagen. The method is simple and does not include any proprietary chemicals or instruments and can be performed on any basic reverse phase UHPLC system with UV detection.

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Creating a Reliable Mass Spectral–Retention Time Library for All Ion Fragmentation-Based Metabolomics

Metabolites ◽

10.3390/metabo9110251 ◽

2019 ◽

Vol 9 (11) ◽

pp. 251 ◽

Cited By ~ 4

Author(s):

Ipputa Tada ◽

Hiroshi Tsugawa ◽

Isabel Meister ◽

Pei Zhang ◽

Rie Shu ◽

...

Keyword(s):

Retention Time ◽

Metabolite Identification ◽

Mass Spectral Library ◽

Spectral Library ◽

Mass Spectral Data ◽

Mass Spectral ◽

Ion Fragmentation ◽

Metabolomics Study ◽

Construction Strategy

Accurate metabolite identification remains one of the primary challenges in a metabolomics study. A reliable chemical spectral library increases the confidence in annotation, and the availability of raw and annotated data in public databases facilitates the transfer of Liquid chromatography coupled to mass spectrometry (LC–MS) methods across laboratories. Here, we illustrate how the combination of MS2 spectra, accurate mass, and retention time can improve the confidence of annotation and provide techniques to create a reliable library for all ion fragmentation (AIF) data with a focus on the characterization of the retention time. The resulting spectral library incorporates information on adducts and in-source fragmentation in AIF data, while noise peaks are effectively minimized through multiple deconvolution processes. We also report the development of the Mass Spectral LIbrary MAnager (MS-LIMA) tool to accelerate library sharing and transfer across laboratories. This library construction strategy improves the confidence in annotation for AIF data in LC–MS-based metabolomics and will facilitate the sharing of retention time and mass spectral data in the metabolomics community.

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Gas Chromatographic Analysis of Barbiturates in Pharmaceuticals

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/51.6.1326 ◽

1968 ◽

Vol 51 (6) ◽

pp. 1326-1329

Author(s):

James L Sibert ◽

Fred L Fricke

Keyword(s):

Retention Time ◽

Chromatographic Analysis ◽

Chromatographic Method ◽

Internal Standard ◽

Gas Chromatographic Analysis ◽

Liquid Chromatographic Method ◽

Liquid Chromatographic

Abstract A rapid gas-liquid chromatographic method is described by which barbiturates can be separated, identified, and quantitated. Two columns are used. A 6′ cyclohexanedimethanol succinate column is used for all barbiturates except phenobarbital, which has an excessively long retention time. Phenobarbital is determined on an 18″ cyclohexanedimethanol succinate column with santonin as an internal standard. A comparison is made between the GLC, USP, and AOAC methods for the analysis of barbiturates.

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Direct glycan structure determination of intact N-linked glycopeptides by low-energy collision-induced dissociation tandem mass spectrometry and predicted spectral library searching

Analytica Chimica Acta ◽

10.1016/j.aca.2016.05.049 ◽

2016 ◽

Vol 934 ◽

pp. 152-162 ◽

Cited By ~ 10

Author(s):

Pei-Jing Pai ◽

Yingwei Hu ◽

Henry Lam

Keyword(s):

Mass Spectrometry ◽

Tandem Mass Spectrometry ◽

Structure Determination ◽

Low Energy ◽

Collision Induced Dissociation ◽

Glycan Structure ◽

Tandem Mass ◽

Energy Collision ◽

Spectral Library

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Standardization of PGC-LC-MS-based glycomics for sample specific glycotyping

10.26434/chemrxiv.7056767.v2 ◽

2019 ◽

Author(s):

Christopher Ashwood ◽

Brian Pratt ◽

Brendan MacLean ◽

Rebekah L. Gundry ◽

Nicolle H. Packer

Keyword(s):

Predictive Models ◽

Tissue Sample ◽

Internal Standard ◽

Search Space ◽

Normalization Method ◽

Porous Graphitized Carbon ◽

Structural Modifications ◽

Graphitized Carbon ◽

Core Fucosylation ◽

Bisecting Glcnac

Porous graphitized carbon (PGC) based chromatography achieves high-resolution separation of glycan structures released from glycoproteins. This approach is especially valuable when resolving structurally similar isomers and for discovery of novel and/or sample-specific glycan structures. However, the implementation of PGC-based separations in glycomics studies has been limited because system-independent retention values have not been established to normalize technical variation. To address this limitation, this study combined the use of hydrolyzed dextran as an internal standard and Skyline software for post-acquisition normalization to reduce retention time and peak area technical variation in PGC-based glycan analyses. This approach allowed assignment of system-independent retention values that are applicable to typical PGC-based glycan separations and supported the construction of a library containing >300 PGC-separated glycan structures with normalized glucose unit (GU) retention values. To enable the automation of this normalization method, a spectral MS/MS library was developed of the dextran ladder, achieving confident discrimination against isomeric glycans. The utility of this approach is demonstrated in two ways. First, to inform the search space for bioinformatically predicted but unobserved glycan structures, predictive models for two structural modifications, core-fucosylation and bisecting GlcNAc, were developed based on the GU library. Second, the applicability of this method for the analysis of complex biological samples is evidenced by the ability to discriminate between cell culture and tissue sample types by the normalized intensity of N-glycan structures alone. Overall, the methods and data described here are expected to support the future development of more automated approaches to glycan identification and quantitation.

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Identification of a Woody-hay Odor-causing Compound in a Drinking Water Supply

Water Science & Technology ◽

10.2166/wst.1999.0309 ◽

1999 ◽

Vol 40 (6) ◽

pp. 273-278 ◽

Cited By ~ 6

Author(s):

Connie C. Young ◽

I. H. (Mel) Suffet ◽

Gil Crozes ◽

August Bruchet

Keyword(s):

Drinking Water ◽

Water Supply ◽

Retention Time ◽

Drinking Water Supply ◽

Spectral Library ◽

Gc Analysis ◽

Grass Hay ◽

Off Flavors ◽

Odor Descriptors

β-Cyclocitral and geosmin have been identified as the chemicals responsible for causing hay/woody and earthy odors in Lake Winnebago, respectively. Initial identifications of off-flavors were made by the GC-MS with an MS spectral library and sensory-GC analysis and the identifications were confirmed by GC retention time and MS spectral matches to the known standard compounds. A Weber-Fechner plot has been developed for β-cyclocitral and its three odor descriptors, fresh grass, hay/woody, and tobacco-like, which were observed at different concentration ranges.

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