Abstract: Protein glycosylation is a common post-translational modification that has significant impacts on protein folding, lifespan, conformation, distribution and function. N-Glycans, which are attached to asparagine residues of proteins, are studied most often due to their compatibility with enzymatic release. Despite the ease of N-glycan release, compositional and structural complexity coupled with poor ionization efficiency during liquid chromatography mass spectrometry (LC-MS) make quantitative glycomic studies a significant challenge. To overcome these challenges, glycans are almost always derivatized prior to LC-MS analyses to impart favorable characteristics, such as improved ionization efficiency, increased LC separation efficiency and the production of more informative fragments during tandem MS. There are a number of derivatization methods available for LC-MS analysis of glycans, each of which imparts different properties that affect both glycan retention on LC columns and MS analyses. To provide guidance for the proper selection of derivatizing reagents and LC columns, herein, we describe a comprehensive assessment of 2-aminobenzamide, procainamide, aminoxyTMT, RapiFluor-MS (RFMS) labeling, reduction and reduction with permethylation for N-glycan analysis. Of the derivatization strategies examined, RFMS provided the highest MS signal enhancement for neutral glycans, while permethylation significantly enhanced the MS intensity and structural stability of sialylated glycans.

Abstract: RATIONALE Within the last decade, applications of compound-specific stable isotope analysis of nitrogen (δ15 N values) in amino acids (CSIA-AA) have developed rapidly, particularly within organismal ecology. Unlike with bulk stable isotope analysis (BSIA), the reproducibility of δ15 N-AA measurements has not been critically assessed. Two primary concerns include the diversity of techniques available for the derivatization of amino acids prior to analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) and the myriad of standardization practices and quality assurance procedures used across studies. METHODS We examined the relative effect of three normalization procedures, (1) internal reference calibration, (2) compound-specific calibration, and (3) scale-normalization, on the accuracy and precision of δ15 N-AA measurements by GC/C/IRMS and the comparability of δ15 N-AA measurements by two derivatization techniques, methoxycarbonylation-esterification and acetylation-esterification, across a range of organisms. RESULTS The overall accuracy and precision of δ15 N-AA measurements were improved following both compound-specific calibration and scale-normalization, as was the comparability of δ15 N-AA measurements of individual amino acids between derivatization techniques across organisms. The mean difference of scale-normalized δ15 N-AA values across all organisms between the two derivatization techniques was 0.19‰, much less than the typical analytical error associated with δ15 N-AA measurements (±1‰). CONCLUSIONS: Adoption of standardized calibration procedures will be important to establishing reproducibility in δ15 N-AA measurements, particularly across derivatization techniques. It is both technically practical and desirable for users of CSIA-AA to adopt practices in quality control and assessment similar to those outlined for BSIA, including the compound-specific calibration of δ15 N-AA values, followed by scale-normalization. Copyright © 2017 John Wiley & Sons, Ltd.

Abstract: Human exposure to polycyclic aromatic hydrocarbons (PAHs) can be assessed through monitoring of urinary mono-hydroxylated PAHs (OH-PAHs) Gas chromatography (GC) has been widely used to separate OH-PAHs before quantification by mass spectrometry in biomonitoring studies However, because GC requires derivatization, it can be time consuming We developed an on-line solid phase extraction coupled to isotope dilution-high performance liquid chromatography-tandem mass spectrometry (on-line-SPE-HPLC-MS/MS) method for the quantification in urine of 1-OH-naphthalene, 2-OH-naphthalene, 2-OH-fluorene, 3-OH-fluorene, 1-OH-phenanthrene, the sum of 2-OH and 3-OH-phenanthrene, 4-OH-phenanthrene, and 1-OH-pyrene The method, which employed a 96-well plate platform and on-line SPE, showed good sensitivity (ie, limits of detection ranged from 0007 to 009 ng/mL) and used only 100 μL of urine Accuracy, calculated from the recovery percentage at three spiking levels, varied from 94 to 113 %, depending on the analyte The inter- and intra-day precision, calculated from 20 repeated measurements of two quality control materials, varied from 52 to 167 % Adequate method performance was also confirmed by acceptable recovery (83–102 %) of two NIST standard reference materials (3672 and 3673) This high-throughput on-line-SPE-HPLC-MS/MS method can be applied in large-scale epidemiological studies

Abstract: Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been used for high-throughput glycan profiling analysis. In spite of the biological importance of sialic acids on nonreducing ends of glycans, it is still difficult to analyze glycans containing sialic acid residues due to their instability and the presence of linkage isomers. In this Article, we describe a one-pot glycan purification/derivatization method employing a newly developed linkage-specific sialic acid derivatization for MS-based glycan profiling with differentiation of sialyl linkage isomer. The derivatization, termed sialic acid linkage specific alkylamidation (SALSA), consists of sequential two-step alkylamidations. As a result of the reactions, α2,6- and α2,3-linked sialic acids are selectively amidated with different length of alkyl chains, allowing distinction of α2,3-/α2,6-linkage isomers from given mass spectra. Our studies using N-glycan standards with known sialyl linkages proved high suitability of SALSA for ...

Abstract: Free fatty acids (FFAs) are vitally important components of lipids that modulate biological metabolism in various ways. Although the molecular structures are simple, the analysis of FFAs is still challenging due to their unique properties and wide concentration range. In the present study, a high-coverage liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was established for the quantification of FFAs in serum samples using two structural analogues 5-(dimethylamino)naphthalene-1-sulfonyl piperazine (Dns-PP) and (diethylamino)naphthalene-1-sulfonyl piperazine (Dens-PP) as twin derivatization reagents. The Dns labeling of FFAs could significantly enhance their MS response via the introduction of the easily ionizable moiety of a tertiary amine-containing part and aid fragmentation in the multiple reaction monitoring (MRM) mode. Our results demonstrated that the detection sensitivities of FFAs were increased by 50–1500 fold compared with the nonderivatization method. At the same time, Dens-label...

Abstract: A synthetic route to 3-formylbenzenesulfonyl chloride derivatives from the corresponding benzaldehydes has been developed. The key step in this procedure is the conversion of aldehyde bisulfite adducts to target compounds via a two-stage reaction in the presence of Na2SO4. A series of 3-formylbenzenesulfonyl chloride derivatives were prepared by this method and identified by chemical derivatization method.

Abstract: Aim: Metabolomics applications represent an emerging field where significant efforts are directed. Derivatization consists prerequisite for GC–MS metabolomics analysis. Methods: Common silylation agents were tested for the derivatization of blood plasma. Optimization of methoxyamination and silylation reactions was performed on a mixture of reference standards, consisting of 46 different metabolites. Stability of derivatized metabolites was tested at 4°C. Results: Optimum results were achieved using N-methyl-N-(trimethylsilyl)trifluoroacetamide. Methoxyamination at room temperature for 24 h followed by 2-h silylation at high temperature lead to efficient derivatization. Conclusion: Formation and stability of derivatives among metabolites differ greatly, so derivatization should be studied before application in metabolomics studies.

Abstract: The cofactor tetrahydrofolate (THF) is used to reduce, oxidize, and transfer one-carbon (1C) units required for the synthesis of nucleotides, glycine, and methionine. Measurement of intracellular THF species is complicated by their chemical instability, signal dilution caused by variable polyglutamation, and the potential for interconversion among these species. Here, we describe a method using negative mode liquid chromatography-mass spectrometry (LC-MS) to measure intracellular folate species from mammalian cells. Application of this method with isotope-labeled substrates revealed abiotic interconversion of THF and methylene-THF, which renders their separate quantitation particularly challenging. Chemical reduction of methylene-THF using deuterated sodium cyanoborohydride traps methylene-THF, which is unstable, as deuterated 5-methyl-THF, which is stable. Together with proper sample handling and LC-MS, this enables effective measurements of five active folate pools (THF, 5-methyl-THF, methylene-THF, methenyl-THF/10-formyl-THF, and 5-formyl-THF) representing the biologically important 1C oxidation states of THF in mammalian cells. Graphical abstract Chemical derivatization with deuterated cyanoborohydride traps unstable methylene-THF as isotope-labeled 5-methyl-THF, enabling accurate quantification by LC-MS.

Abstract: Derivatization is the most powerful contribution to the identification and quantification of plant cannabinoids (p-CBDs) by gas chromatography-mass spectrometry (GC-MS): providing volatile derivatives with eminent properties (high selectivity, outstanding sensitivity and mass spectrometric peculiarities). These derivatives are excellent candidates to determine the main p-CBDs, like tetrahydrocannabivarin (THCV), tetrahydrocannabidivarin (CBDV), cannabidiol (CBD), cannabichromene (CBC), cannabicyclol (CBCL), tetrahydrocannabinol (THC), cannabigerol (CBG), cannabinol (CBN), 11-hydroxy-Δ 9 -THC (11-OH-THC) and 11-nor-9-carboxy-THC (THC-COOH). Identification and quantification of p-CBDs is required – partly as trace constituents in complex biological matrices of drug users, partly as main components in seizure samples: in both cases, in extremely different ratios. GC proposals published between 2000 and 2017, along with outstanding pioneer contributions, are reviewed. Procedures, without derivatization and applying various alkylsilyl, acylation and/or esterification techniques were listed, compared and criticized. Further sorting was based on the reagent type, on examined matrices, on enrichment/detection related acquisition protocols and on analytical performance characteristics.

Abstract: The reaction of LGa (L = Dipp(4-(Dipp-imino)pent-2-en-2-yl)amide; Dipp: 2,6-diisopropylphenyl) and white phosphorus was revisited. A plethora of unprecedented polyphosphanes in addition to the known monoinserted product LGaP4 (1) are observed. An optimized synthesis of the hitherto unknown hexaphosphane (LGa)2P6 (3) is presented, and its subsequent selective derivatization with Bronsted acids, MeOTf, Ph2ECl (E = P, As), and NaOCP provides access to a wealth of functionalized hexa- and heptaphosphanes.

Abstract: Ion chromatography coupled to a quadrupole Orbitrap mass analyzer was used to develop a multiresidue method for the determination of highly polar pesticides and their metabolites (chlorate, perchlorate, fosetyl-aluminum, glyphosate, aminomethylphosphonic acid (AMPA), phosphonic acid, N-acetyl AMPA, and N-acetyl glyphosate) in fruits and vegetables. After extraction with methanol, samples were diluted 5× with water. No derivatization was applied. Pesticides were separated in an anion-exchange column. Water was used as the ion chromatography mobile phase. A gradient was created by increasing the concentration of KOH in the mobile phase. Ion chromatography provided good and stable retention and separation for all studied compounds. All investigated pesticides had an LOQ of 0.01 mg/kg and a linear range of 0.01-0.50 mg/kg. The ion ratio of the m/z ions produced was stable and adequate (deviation 100 000) provided by the Orbitrap analyzer with the low m/z ions obtained (e.g., m/z 80) was effective in obtaining low background matrix signals. The influence of postcolumn infusion of organic solvent on sensitivity was investigated. Acetonitrile was found to be more effective than methanol, increasing the sensitivity 3× with respect to water. The method was validated for five vegetable-based matrixes. Both the sample processing and the analytical measurement were very fast. Hence, the methodology is ideal for high-throughput work.

Abstract: Lignin and humins are both (by-)products of biorefining processes aimed at the valorization of lignocellulosic biomass. In order to improve the efficiency of such biorefineries and to develop new valorization pathways for these polymeric materials, detailed insight into their complex chemical structure and functional group distribution is required. Here, we report on the quantification and classification of the ketone and aldehyde carbonyl functional groups contained in these two polymers by 19F NMR. The known method of carbonyl derivatization with 4-(trifluoromethyl)phenylhydrazine to the corresponding hydrazone has been improved and simplified, allowing derivatization directly in an NMR tube, requiring no additional workup before quantification by 19F NMR. Furthermore, the scope of the method was assessed and expanded, with model compound studies, which included monomeric and dimeric compounds as well as synthetic polymers, showing that the carbonyl functions can indeed be reproducibly quantified. Using...

Abstract: Amino acids play a key role in food analysis, clinical diagnostics, and biochemical research. Capillary electrophoresis with laser-induced fluorescence detection was used for the analysis of several amino acids. Amino acid labeling with fluorescein isothiocyanate was conducted using microwave-assisted derivatization at 80 degrees C (680 W) during only 150 s. Good electrophoretic resolution was obtained using a background electrolyte composed of sodium tetraborate buffer (100 mM; pH 9.4) and $\beta$-cyclodextrin (10 mM), and the limits of quantification were 3-30 nM. The developed capillary electrophoresis with laser-induced fluorescence method was used to analyze amino acids in $Dunaliella\ salina$ green algae grown under different conditions. A simple extraction technique based on electroporation of the cell membrane was introduced. A home-made apparatus allowed the application of direct and alternating voltages across the electrochemical compartment containing a suspension of microalgae in distilled water at 2.5 g/L$^{-1}$. A direct voltage of 12 V applied for 4 min gave the optimum extraction yield. Results were comparable to those obtained with accelerated-solvent extraction. The efficiency of electroporation in destroying microalgae membranes was shown by examining the algae surface morphology using scanning electron microscopy. Stress conditions were found to induce the production of amino acids in $Dunaliella\ salina$ cells.

Abstract: In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable ...

Abstract: Small molecules containing carboxylic acid functional groups are ubiquitous throughout biology, playing vital roles in biological chemistry ranging from energy metabolism to cellular signaling. This paper describes a new derivatization reagent, 4-bromo-N-methylbenzylamine, which was selected for its potential to derivatize mono-, di- and tri-carboxylic acids, such as the intermediates of the tricarboxylic acid (TCA) cycle. This derivatization procedure facilitated the use of positive electrospray ionization (ESI) tandem mass spectrometry (MS/MS) detection of derivatized species allowing for clear identification thanks to the easily recognizable isotope pattern of the incorporated bromine. A liquid chromatography (LC)-MS/MS method was developed which provided limits of detection between 0.2 and 44 μg L−1 in under 6 min, depending on the analyte and total analysis time. This method was successfully applied in both in vitro and in vivo models.

Abstract: An effective method based on automated online headspace solid-phase microextraction (HS-SPME) followed by on-fiber derivatization coupled with gas chromatography-mass spectrometry (GC-MS) for simultaneous determination of eleven estrogenic and odorous chloro- and bromo-phenolic compounds was developed. The target chemicals included seven chlorophenols (CPs): 2-chlorophenol (2-CP), 4-chlorophenol (4-CP), 2-chloro-4-methyl phenol (2-C-4-MP), 4-chloro-2-methyl phenol (4-C-2-MP), 4-chloro-3-methyl phenol (4-C-3-MP), 2,6-dichlorophenol (2,6-DCP), and 2,4,5-trichlorophenol (2,4,5-TCP) and four bromophenols (BPs): 2-bromophenol (2-BP), 2,6-dichlorophenol (2,6-DBP), 2,4-dichlorophenol (2,4-DBP), and 2,4,6-tribromophenol (2,4,6-TBP). The extraction temperature, extraction time, derivatization time, salt concentration and pH of sample solution were optimized and determined in this study. The developed method using the optimized conditions was validated with respect to the linearity (R2 > 0.991), low limit of detection (LOD, 8.8–42.9 ng L−1), and excellent recoveries (69–108%) with a low relative standard deviation (RSD, 1.5–18.6%). This method provides an effective online extraction and derivatization process with good sensitivity in GC-MS, and can be applied for the efficient determination of chloro- and bromo-phenolic compounds in surface water.