Approaches to liquid chromatography tandem mass spectrometry assessment of glyphosate residues in wine

The performance of two different analytical methodologies to investigate the presence of glyphosate (GLY) and aminomethylphosphonic acid (AMPA) residues in wine samples was evaluated. Transformation of compounds in their fluorene-9-methyloxycarbonyl derivatives permitted their separation under reversed-phase liquid chromatography with tandem mass spectrometry (LC–MS/MS) determination. Although the wine matrix severely impaired the efficiency of GLY derivatization, this drawback was solved using a molecularly imprinted sorbent for the previous, selective extraction of GLY and AMPA from wine. Alternatively, the use of a strong anionic exchange, polyvinyl alcohol-based LC column, turned to be the most effective alternative for direct determination of both compounds in diluted wine samples. The chromatographic behavior of this column and the magnitude of matrix effects observed during analysis of diluted wine samples were significantly affected by the composition of the mobile phase. Under final working conditions, this column permitted the separation of AMPA and the fungicide fosetyl (which shows common transitions in tandem MS/MS methods), it improved significantly the sample throughput versus extraction-derivatization-purification method, and it allowed the use of solvent-based calibration standards. Both analytical procedures provided similar limits of quantification (LOQs) for GLY (0.5–1.0 ng mL−1), while the multistep method was 8 times more sensitive to AMPA than the direct procedure. GLY residues stayed above method LOQs in 70% of the processed wines; however, concentrations measured in 95% of positive samples remained 100 times below the maximum residue limit (MRL) set for GLY in vinification grapes. Graphical abstract

Abnormal retention of s-triazine herbicides on porous graphitic carbon

Porous graphitic carbon (PGC) is a widely used stationary phase for reversed-phase high-performance liquid chromatography (HPLC) that allows separation of structurally similar compounds retained in mixed form on a flat graphite surface. Such a stationary phase can be used in analytical chemistry to provide good separation and selectivity in pesticide monitoring. In this article, we studied the chromatographic behavior of five common triazine herbicides (simazine, atrazine, desmetryn, propazine, prometryn) on PGC vis-à-vis octadecyl-functionalized silica gel (ODS). It was found that the herbicides studied have an abnormal elution order on PGC compared to ODS. PGC was also characterized by higher selectivity of analyte separation. This behavior of triazine herbicides on PGC cannot be explained either with the help of existing theory or by mathematical modeling of adsorption processes on graphite. Therefore, we have proposed a possible retention mechanism, explaining the effects observed, due to the shielding of the amino group in the triazine ring by alkyl substituents, which decreases the “polar retention effect” of PGC. Satisfactory separation efficacy was obtained with the proposed analytical method, using convenient UV-detection and without resort to laborious techniques such as HPLC coupled with mass spectrometry.

Rapid Separation of Human Hemoglobin on a Large Scale From Non-clarified Bacterial Cell Homogenates Using Molecularly Imprinted Composite Cryogels

The production of a macroporous hydrogel column, known as cryogel, has been scaled up (up to 150 mL) in this work for the purification of human hemoglobin from non-clarified bacterial homogenates. Composite cryogels were synthesized in the presence of adult hemoglobin (HbA) to form a molecularly imprinted polymer (MIP)network where the affinity sites for the targeted molecule were placed directly on an acrylamide cryogel by protein imprinting during the cryogelation. The MIP composite cryogel column was first evaluated in a well-defined protein mixture. It showed high selectivity toward HbA in spite of the presence of serum albumin. Also, when examined in complex non-clarified E. coli cell homogenates, the column showed excellent chromatographic behavior. The binding capacity of a 50 mL column was thus found to be 0.88 and 1.2 mg/g, from a protein mixture and non-clarified cell homogenate suspension, respectively. The recovery and purification of the 50 mL column for separation of HbA from cell suspension were evaluated to be 79 and 58%, respectively. The MIP affinity cryogel also displayed binding and selectivity toward fetal Hb (HbF) under the same operational conditions.

Systematic Characterization and Identification of Saikosaponins in Extracts From Bupleurum marginatum var. stenophyllum Using UPLC-PDA-Q/TOF-MS

Saikosaponins comprise a large group of chemical components present in the Bupleurum species that have attracted attention in the field of medicine because of their significant biological activities. Due to the high polarity, structural similarity, and the presence of several isomers of this class of components, their structural identification is extremely challenging. In this study, the mass spectrometric fragmentation pathways, UV spectral features, and chromatographic behavior of different types of saikosaponins were investigated using 24 standard substances. Saikosaponins containing carbonyl groups (C=O) in the aglycone produced fragment ions by loss of 30 Da, and in addition, type IV saikosaponins could produce [aglycone−CH2OH−OH−H]− and [aglycone−H2O−H]− fragment ions through neutral losses at positions C16 and C17. The above characteristic ions can be used to identify saikosaponins. More notably, the identification process of saikosaponins was systematically summarized, and using this method, 109 saikosaponins were identified or tentatively characterized from the saikosaponins extract of Bupleurum marginatum var. stenophyllum (BMS) using UPLC-PDA-Q/TOF-MS with both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes, of which 25 were new compounds and 60 were first discovered from BMS. Further studies revealed that the saikosaponins profiles of BMS, Bupleurum chinense DC (BC), and Bupleurum marginatum Wall. ex DC (BMW) were very similar. This work is of great significance for the basic research of the Bupleurum species and provides strong technical support to solve the resource problems associated with Radix Bupleuri.

Discovery of Signature Cyclic Immonium Ion for Lactyllysine Reveals Widespread and Functional Lactylation on Non-histone Proteins

Lactylation is a new modification discovered on histones. However, whether it can be installed on non-histone proteins remains unclear. Here we report the formation of a signature cyclic immonium ion of lactyllysine, together with the characteristically changed chromatographic behavior, enabling confident protein lactylation assignment by mass spectrometry. This identification strategy was confirmed by affinity-enriched lactylation proteome and revealed lactylation on nuclear non-histone proteins such as nucleolin. Subsequent exploitation of the approach to mining unenriched, deep proteome resources unveiled an understudied lactylation landscape. From the draft map of the Human Proteome, we identified widespread lactylation on DHRS7 among human tissues, and demonstrated site-directed mutagenesis of the lactylated site affects previously unannotated proteinaceous association. Additionally, the Meltome Atlas showed lactylation frequently occurs on glycolytic enzymes and concomitantly induces thermal stability changes on carrier enzymes. Collectively, the identified signatures of protein lactylation enable confident assignment and allow for the discovery of lactylation proteome expanding beyond histones, representing a step to further understand how lactylation governs cells.

Development of Theoretical Approaches to Determination of the Main Groups of Biologically Active Substances of Medicinal Plant Raw Materials by TLC Method

Introduction. As is known, the selection of optimal conditions for the analysis of extracts from medicinal plant raw materials (MPМ), as well as medicinal herbal preparations and pharmaceutical substances of plant origin, characterized by a complex variable composition of biologically active substances (BAS) by TLC, presents certain difficulties. For the design of mobile phases for the separation of mixtures of BAS of plant origin, in a thin layer of sorbent, the following approaches are used: literary sources; standard mobile phases; spot elution method; the scheme proposed by the firm Camag (Switzerland); model "PRISMA"; variocameras and others. In foreign literature, there are publications on the generalization of the available experimental data on the determination of various natural groups of BAS in objects of plant origin. However, such reviews did not reveal the regularities of the chromatographic behavior of individual BAS in a thin layer, as well as the influence of various factors on the reproducibility of R f values. The study of the possibility of a theoretical approach to the choice of optimal conditions for chromatography of groups of BAS of different polarity, allowing them to separate, identify and quantify by TLC is a relevant and poorly developed area of chromatography in general.Aim. The aim of this work was to develop a theoretical approach to the choice of optimal conditions for the chromatographic separation of various groups of BAS of plant origin in a thin layer of sorbent.Materials and methods. To study the regularities of chromatographic behavior in a thin layer of representatives of the main classes of BAS present in MPМ (amino acids, flavonoids, tannins, simple sugars, ascorbic acid, fat-soluble vitamins), the value of the main factor affecting the parameters of the efficiency of the chromatographic process, the polarity of the eluent, was studied. As objects of research, we used ready-made chopped raw material of nettle leaves, produced by a domestic manufacturer, that meets the requirements of regulatory documents, as well as sea buckthorn fruits collected on the territory of the Voronezh region, according to the rules for harvesting MPМ of various morphological groups in fresh and dried form.Results and discussion. The regularities of elution and mathematical models describing the chromatographic behavior of plant BAS in a thin layer of sorbent have been established. Based on the totality of the results obtained, from the standpoint of the efficiency of the chromatographic process, the optimal conditions for their TLC analysis were selected and theoretically substantiated. To study the qualitative composition of BAS and to achieve a clear separation of zones on chromatograms, TLC methods were developed and tested on the studied MPМ using simple, frontal or two-dimensional chromatography.Conclusion. It is shown that the determination and separation in a thin layer of the sorbent of hydrophilic and lipophilic BAS of MPМ in the presence of a joint requires different approaches and techniques. The paper proposes an algorithm for the selection of the mobile phase and methods of chromatography of BAS of medicinal products. The revealed mathematical models describing the chromatographic behavior of BAS will make it possible to select the conditions under which it is possible to determine the individual components of multicomponent mixtures without preliminary separation. The developed methods for the determination of BAS can also be used for standardization and quality assessment of other types of MPМ, phytopreparations and pharmaceutical substances of plant origin.

Predicting Pharmacokinetic Properties of Potential Anticancer Agents via Their Chromatographic Behavior on Different Reversed Phase Materials

The Quantitative Structure-Activity Relationship (QSAR) methodology was used to predict biological properties, i.e., the blood–brain distribution (log BB), fraction unbounded in the brain (fu,brain), water-skin permeation (log Kp), binding to human plasma proteins (log Ka,HSA), and intestinal permeability (Caco-2), for three classes of fused azaisocytosine-containing congeners that were considered and tested as promising drug candidates. The compounds were characterized by lipophilic, structural, and electronic descriptors, i.e., chromatographic retention, topological polar surface area, polarizability, and molecular weight. Different reversed-phase liquid chromatography techniques were used to determine the chromatographic lipophilicity of the compounds that were tested, i.e., micellar liquid chromatography (MLC) with the ODS-2 column and polyoxyethylene lauryl ether (Brij 35) as the effluent component, an immobilized artificial membrane (IAM) chromatography with phosphatidylcholine column (IAM.PC.DD2) and chromatography with end-capped octadecylsilyl (ODS) column using aqueous solutions of acetonitrile as the mobile phases. Using multiple linear regression, we derived the statistically significant quantitative structure-activity relationships. All these QSAR equations were validated and were found to be very good. The investigations highlight the significance and possibilities of liquid chromatographic techniques with three different reversed-phase materials and QSARs methods in predicting the pharmacokinetic properties of our important organic compounds and reducing unethical animal testing.

Estimation of lipophilicity and design of new 17β-carboxamide glucocorticoids using RP-HPLC and quantitative structure-retention relationships analysis

Eight 17β-carboxamide glucocorticoids with local anti-inflammatory activity were selected and their retention behavior tested in six RP-HPLC systems (I–VI). logkw, a, and φ0 parameters were calculated and correlation with previously determined logPo/w values was examined. RP-HPLC system IV, which consisted of cyano column and methanol–water mobile phases (50:50, 60:40, 70:30, and 80:20, v/v), was selected as the most reliable for lipophilicity prediction and used for the analysis of chromatographic behavior of remaining fourteen 17β-carboxamide glucocorticoids. Quantitative structure-retention relationships analysis was performed and PLS(logkw) model was selected as the most statistically significant. On the basis of selected model and interpretation of corresponding descriptors, new derivatives with higher logkw values and higher expected lipophilicity were designed.

Vitamin D analyses in veterinary feeds by gas chromatography-tandem mass spectrometry

This report examines the feasibility of determination of Vitamin D3, D2 and their 25-hydroxy metabolites utilizing Gas Chromatography Tandem Mass Spectrometry (GC/MS/MS) as a potential alternative to popular Liquid Chromatography Tandem Mass Spectrometric (LC/MS/MS) methodologies. The GC/MS/MS approach was found to operate reasonably well despite long-standing concerns that gas-liquid chromatography of vitamin D compounds invoke thermal rearrangements owing to the relatively high inlet and capillary column temperatures used. The workup procedure involved incubation of feed samples with concentrated potassium hydroxide for overnight fat saponification, extraction of D Vitamins in n-hexane and reaction with N,O-bis(trimethylsilyl)trifluoroacetamide at 70 °C for 30 mins. In addition to parent compounds, small amounts of pyro-, isopyro-, and iso-vitamin D and isotachysterol3 variants were obtained from each Vitamin D-related compound upon extraction and GC/MS/MS analysis. Mass spectral and chromatographic behavior of these compounds are herein described and interpreted. Multiple Reaction Monitoring settings on GC/MS/MS included m/z 456→351 for Vitamin D3 and m/z 486→363 for Vitamin D2. Trimethylsilylation enabled single predominant peaks for Vitamins D3 and D2, and sample workup in the presence of deuterated Vitamin D analogs enabled accurate and precise sensitivity to 1 ppb (ng/g) in feeds. The method could be extended with reasonable accuracy to 25-hydroxy (25OH) compounds, but accuracies would be significantly improved by inclusion of respective 25OH-specific deuterated internal standards. The method was applied to 27 submissions of suspect dog foods of which 22% were discovered elevated and 44% were discovered to contain toxic levels of Vitamin D3. The described method was thus discovered to provide a suitable mass spectrometric approach for Vitamin D, proving itself here specifically of value in detection of ergocalciferol and cholecalciferol in animal feeds. The specificity and sensitivity of the tandem quadrupole approach can enable suitable applicability to serum determination if desired.