Reversed-phase Liquid Chromatographic Separation Research Articles

The identification of psychedelic compounds within Australian Acacia species

ABSTRACT N,N-dimethyltryptamine (DMT) is a psychedelic substance produced within various Acacia species; however, the range of species that contain it and the quantity of the compound found within each species is relatively unknown. This work focused on the identification of DMT in 104 plant samples, representing 79 Acacia species, utilizing a reverse phase liquid chromatographic separation prior to mass spectral characterization with a robust Q-TOF-MS method. The findings of this work determined 51 DMT-containing species, 41 of which were previously unknown to contain the psychedelic.

The Impact of Different Drying Methods on the Metabolomic and Lipidomic Profiles of Arthrospira platensis.

Drying is an inseparable part of industrial microalgae production. In this work, the impacts of eight different drying methods on the metabolome and lipidome of Arthrospira platensis were investigated. The studied drying methods were freeze drying (FD), sun drying (SD), air drying at 40 and 75 °C (AD' and AD″), infrared drying at 40 and 75 °C (IRD' and IRD″), and vacuum drying at 40 and 75 °C (VD' and VD″). Results gathered by reversed-phase liquid chromatography separation coupled with high-resolution tandem mass spectrometry with electrospray ionization (RP-LC-ESI-Orbitrap HRMS/MS) analysis allowed researchers to identify a total of 316 metabolites (including lipids) in aqueous and ethanolic extracts. The compounds identified in ethanolic extracts were mainly lipids, such as neutral and polar lipids, chlorophylls and carotenoids, while the compounds identified in the aqueous extracts were mainly amino acids and dipeptides. Among the identified compounds, products of enzymatic and chemical degradation, such as pyropheophytins, monoacylglycerols and lysophosphatidylcholines were also identified and their amounts depended on the drying method. The results showed that except for FD method, recognized as a control, the most protective method was AD'. Contrary to this, VD' and VD″, under the conditions used, promoted the most intense degradation of valuable metabolites.

Untargeted metabolomics reveals potential plasma biomarkers for diagnosis of primary aldosteronism using liquid chromatography-mass spectrometry.

Metabolite profiling has the potential to comprehensively bridge phenotypes and complex heterogeneous physiological and pathological states. We performed a metabolomics study using parallel liquid chromatography-mass spectrometry (LC-MS) combined with multivariate data analysis to screen for biomarkers of primary aldosteronism (PA) from a cohort of 111 PA patients and 218 primary hypertension (PH) patients. Hydrophilic interaction chromatography and reversed-phase liquid chromatography separations were employed to obtain a global plasma metabolome of endogenous metabolites. The satisfactory classification between PA and PH patients was obtained using the MVDA model. A total of 35 differential metabolites were screened out and identified. A diagnostic biomarker panel was established using the least absolute shrinkage and selection operator (LASSO) binary logistic regression model and receiver operating characteristic analysis. Joint analysis with clinical indicators, including plasma supine aldosterone level, plasma orthostatic aldosterone level, body mass index, and blood potassium, revealed that the combination of metabolite biomarker panel and plasma supine aldosterone has the best clinical diagnostic efficacy.

Temperature-responsive comprehensive two-dimensional liquid chromatography coupled to high resolution mass spectrometry for the elucidation of the oxidative degradation processes of chemicals of environmental concern

This study explores the possibilities offered by temperature-responsive liquid chromatography (TRLC) based comprehensive 2-dimensional liquid chromatography in combination with reversed-phase liquid chromatography (RPLC) for the analysis of degradation products formed upon oxidative treatment of persistent organic pollutants, in this case exemplified through carbamazepine (CBZ). The TRLC×RPLC combination offers the possibility to overcome peak overlap and incomplete separation encountered in 1D approaches, while the transfer of the purely aqueous mobile phase leads to refocusing of all analytes on the second dimension column. Consequently, this allows for about method-development free and hence, easier LC×LC. The study focuses on the oxidative degradation of CBZ, a compound of environmental concern due to its persistence in water bodies. The TRLC×RPLC combination effectively separates and identifies CBZ and its degradation products, while offering improved selectivity over the individual TRLC or RPLC separations. This allows gathering more understanding of the degradation cascade and allows real-time monitoring of the appearance and disappearance of various degradation products. The compatibility with high-resolution mass spectrometry is last shown, enabling identification of 21 CBZ-related products, nine of which were not previously reported in CBZ degradation studies. The approach's simplicity, optimization-free aspects, and ease of use make it a promising tool for the analysis of degradation pathways in environmental contaminants.

Monosaccharide Analysis After One-Pot Derivatization Followed by Reverse-Phase Liquid Chromatography Separation and UV/Vis Detection.

Derivatization of monosaccharides with 1-phenyl-3-methyl-5-pyrazolone (PMP) introduces two chromophores per sugar molecule. Their separation on a superficially porous C18 reverse-phase column, using common liquid chromatography equipment, results in short analysis times (under 20min) and high sensitivity (limit of quantitation 1nmol). This method allows for complex monosaccharide mixtures to be separated and quantified using a reasonably simple and safe derivatization procedure.

An optimized methodology for the determination of multiclass organic ultraviolet sunscreens and metabolites in human milk through chromatographic and chemometric resolution

Organic UV filters (UVFS) are used to mitigate the dermal effects associated with health risks from UV radiation, making them essential in personal care products. UVFS are frequently identified in environmental samples due to their high lipophilicity and persistence, underscoring the urgency of comprehensive assessments and regulatory measures aimed at safeguarding ecosystems and human health. The present study reports a multiclass analytical method for determining 16 UV sunscreens and metabolites in breast milk based on an ultrasound-assisted-dispersive liquid-liquid micro-extraction (UA-DLLME) with further chromatographic and chemometric resolution. The experimental conditions of the UA-DLLME were optimized through the implementation of the Design of Experiment tools. To model the responses, least-squares and artificial neural network methodologies were implemented. The optimal conditions were found by employing the desirability function. The samples were analyzed through reverse-phase liquid chromatographic separation, UV diode array, and fast-scanning fluorescence detection. The chromatographic analysis enabled the resolution of 16 analytes in a total time of 13.0 min. Multivariate curve resolution-alternating least-square (MCR-ALS) modelling was implemented to resolve analytes that were not fully resolved and to determine analytes that coeluted with endogenous components of the breast milk samples. An enrichment factor of 5-fold concentration was obtained with this methodology, reaching recoveries between 65 % and 105 % for 13 multiclass UV sunscreens and metabolites in breast milk samples with RSD % and REP % lower than 12 %.

Inclusion of Porous Graphitic Carbon Chromatography Yields Greater Protein Identification and Compartment and Process Coverage and Enables More Reflective Protein-Level Label-Free Quantitation.

The ubiquity of mass spectrometry-based bottom-up proteomic analyses as a component of biological investigation mandates the validation of methodologies that increase acquisition efficiency, improve sample coverage, and enhance profiling depth. Chromatographic separation is often ignored as an area of potential improvement, with most analyses relying on traditional reversed-phase liquid chromatography (RPLC); this consistent reliance on a single chromatographic paradigm fundamentally limits our view of the observable proteome. Herein, we build upon early reports and validate porous graphitic carbon chromatography (PGC) as a facile means to substantially enhance proteomic coverage without changes to sample preparation, instrument configuration, or acquisition methods. Analysis of offline fractionated cell line digests using both separations revealed an increase in peptide and protein identifications by 43% and 24%, respectively. Increased identifications provided more comprehensive coverage of cellular components and biological processes independent of protein abundance, highlighting the substantial quantity of proteomic information that may go undetected in standard analyses. We further utilize these data to reveal that label-free quantitative analyses using RPLC separations alone may not be reflective of actual protein constituency. Together, these data highlight the value and comprehension offered through PGC-MS proteomic analyses. RAW proteomic data have been uploaded to the MassIVE repository with the primary accession code MSV000091495.

“Ghost peaks” in reversed-phase liquid chromatography separation of an electron-rich aniline compound: Mechanism and solution for this phenomenon

Two “ghost peaks” appeared in the process of developing an impurities analytical method for an electron-rich aniline compound by RP-HPLC. Surprisingly, the “ghost peaks” disappeared when the mobile phase was re-prepared. The "ghost peak" slowly began to reappear and gradually increased after increasing the injection volume or changing different instruments and columns. In this paper, the structures of two “ghost peaks” were analyzed by LC-MS/MS, which were identified as the dimer of the main peak and the oxidation products of the dimer. The mechanism was confirmed by studying the “ghost peaks” appearance phenomenon in the mobile phase with different pH values, different types of buffer salts, and different brand chromatographic columns. The potential chemical behavior of this type of compound on the column was clearly revealed in the process of RP-HPLC separation. The coupling reaction of electron-rich aniline compounds can be induced by partial or complete dissociation of the silanol group to Si-O−in the stationary phase of a reversed-phase chromatographic column under neutral and basic conditions of the mobile phase. Adding inorganic salts to the mobile phase can inhibit the reaction, and the higher salt concentration, the stronger the effect.

GeLC-FAIMS-MS workflow for in-depth middle-down proteomics.

Middle-down proteomics (MDP) is an analytical approach in which protein samples are digested with proteases such as Glu-C to generate large peptides (>3kDa) that are analyzed by mass spectrometry (MS). This method is useful for characterizing high-molecular-weight proteins that are difficult to detect by top-down proteomics (TDP), in which intact proteins are analyzed by MS. In this study, we applied GeLC-FAIMS-MS, a multidimensional separation workflow that combines gel-based prefractionation with LC-FAIMS MS, for deep MDP. Middle-down peptides generated by optimized limited Glu-C digestion conditions were first size-fractionated by polyacrylamide gel electrophoresis, followed by C4 reversed-phase liquid chromatography separation and additional ion mobility fractionation, resulting in a significant increase in peptide length detectable by MS. In addition to global analysis, the GeLC-FAIMS-MS concept can also be applied to targeted MDP, where only proteins in the desired molecular weight range are gel-fractionated and their Glu-C digestion products are analyzed, as demonstrated by targeted analysis of integrins in exosomes. In-depth MDP achieved by global and targeted GeLC-FAIMS-MS supports the exploration of proteoform information not covered by conventional TDP by increasing the number of detectable protein groups or post-translational modifications (PTMs) and improving the sequence coverage.

Analysis of Sialic Acid Linkage in N-Linked Glycopeptides Using Liquid Chromatography-Electron-Activated Dissociation Time-of-Flight Mass Spectrometry.

Herein, we report a novel liquid chromatography coupled with tandem mass spectrometry method to characterize N-acetylneuraminic acid (Neu5Ac, Sa) linkage in N-linked glycans in glycopeptides with no sialic acid derivatization. First, we established a separation in reversed-phase high-performance liquid chromatography (HPLC) using a higher formic acid concentration in the mobile phases, which separated the N-glycopeptides depending on the Sa linkage. We also demonstrated a novel characterization method of Sa linkages in N-glycopeptides using electron-activated dissociation. We found that hot electron capture dissociation using an electron beam energy higher than 5 eV cleaved glycosidic bonds in glycopeptides, resulting in each glycosidic bond in the antennas being broken on both sides of the oxygen atom. Such glycosidic bond cleavage at the reducing end (C-type ion) showed the difference in Sa linkages between Sa-Gal, Gal-GlcNAc, and GlcNAc-Man. We proposed a rule to characterize the Sa linkages using the Sa-Gal products. This method was applied to N-glycopeptides in tryptic fetuin digest separated by an optimized reversed-phase HPLC. We successfully identified a number of isomeric glycoforms in the glycopeptides with different Sa links, whose peptide backbones were also simultaneously sequenced by hot ECD.

Design of β-Alanine-Derived Novel C18 Stationary Phase Containing Embedded Urea and Amide Groups for the Separation of Versatile Analytes.

Novel stationary phases have been emerging recently. A β-alanine-derived embedded urea and amide group-containing C18 phase (Sil-Ala-C18) was prepared for the first time. The media were packed into a 150 × 2.1 mm HPLC column, and the newly designed column was evaluated with the Tanaka and Neue test protocols in reversed-phase liquid chromatography (RPLC) separation mode. Moreover, it was characterized by the Tanaka test protocol in hydrophilic interaction chromatography (HILIC) separation mode. The new phase was characterized by elemental analysis, attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA), and solid-state 13C cross-polarization magic angle spinning (CP/MAS) NMR spectroscopy at variable temperatures. The chromatographic evaluation involved very good separation of nonpolar shape-constrained isomers, polar and basic compounds in RPLC, and highly polar compounds in HILIC compared to the commercial reference columns. The Sil-Ala-C18 phase was able to separate the challenging β- and γ-isomers of tocopherol. The phase was also successfully applied for the separation of the isomers of tocopherol (vitamin E) and capsaicinoids from real samples of chili peppers (Capsicum spp.) in RPLC and ascorbic acid (vitamin C) in HILIC.

Identification of proteoforms by top-down proteomics using two-dimensional low/low pH reversed-phase liquid chromatography-mass spectrometry.

In top-down (TD) proteomics, efficient proteoform separation is crucial to reduce the sample complexity and increase the depth of the analysis. Here, we developed a two-dimensional low pH/low pH reversed-phase liquid chromatography separation scheme for TD proteomics. The first dimension for offline fractionation was performed using a polymeric reversed-phase (PLRP-S) column with trifluoroacetic acid as ion-pairing reagent. The second dimension, a C4 nanocolumn with formic acid as ion-pairing reagent, was coupled online with a high-field asymmetric ion mobility spectrometry (FAIMS) Orbitrap Tribrid mass spectrometer. For both dimensions several parameters were optimized, such as the adaption of the LC gradients in the second dimension according to the elution time (i.e., fraction number) in the first dimension. Avoidance of elevated temperatures and prolonged exposure to acidic conditions minimized cleavage of acid labile aspartate-proline peptide bonds. Furthermore, a concatenation strategy was developed to reduce the total measurement time. We compared our low/low pH with a previously published high pH (C4, ammonium formate)/low pH strategy and found that both separation strategies led to complementary proteoform identifications, mainly below 20kDa, with a higher number of proteoforms identified by the low/low pH separation. With the optimized separation scheme, more than 4900 proteoforms from 1250 protein groups were identified in Caco-2 cells.

Reversed-Phase Liquid Chromatography of Peptides for Bottom-Up Proteomics: A Tutorial.

The performance of the current bottom-up liquid chromatography hyphenated with mass spectrometry (LC-MS) analyses has undoubtedly been fueled by spectacular progress in mass spectrometry. It is thus not surprising that the MS instrument attracts the most attention during LC-MS method development, whereas optimizing conditions for peptide separation using reversed-phase liquid chromatography (RPLC) remains somewhat in its shadow. Consequently, the wisdom of the fundaments of chromatography is slowly vanishing from some laboratories. However, the full potential of advanced MS instruments cannot be achieved without highly efficient RPLC. This is impossible to attain without understanding fundamental processes in the chromatographic system and the properties of peptides important for their chromatographic behavior. We wrote this tutorial intending to give practitioners an overview of critical aspects of peptide separation using RPLC to facilitate setting the LC parameters so that they can leverage the full capabilities of their MS instruments. After briefly introducing the gradient separation of peptides, we discuss their properties that affect the quality of LC-MS chromatograms the most. Next, we address the in-column and extra-column broadening. The last section is devoted to key parameters of LC-MS methods. We also extracted trends in practice from recent bottom-up proteomics studies and correlated them with the current knowledge on peptide RPLC separation.

Positional Assignment of C-C Double Bonds in Fatty Acyl Chains of Intact Arsenosugar Phospholipids Occurring in Seaweed Extracts by Epoxidation Reactions.

Water-soluble diacyl arsenosugar phospholipids (As-PL) are natural products widespread in marine animals and algae, including the brown alga Undaria pinnatifida, also known as wakame. The systematic recognition of As-PL has been hampered by the lack of standard and of qualitative methods to establish the carbon-carbon double bond positions of unsaturated fatty acyl chains. Here, the epoxidation reaction of fatty acyl substituents of As-PL was carried out with high selectivity by meta-chloroperoxybenzoic acid and the C-C double bond localization was established by collision-induced dissociation of epoxidized species as deprotonated molecules, [epoM - H]-. Reversed-phase liquid chromatography (RPLC) separation and a sequential triple-stage MS (i.e., MS3) analysis of unsaturated and epoxidized As-PL were very helpful to characterize the carbon-carbon double bond locations of both sn-1 and sn-2 fatty acyl chains, starting from a diagnostic product ion pair with 16.0 Da mass difference. These results indicate that intact As-PL can be annotated in terms of fatty acyl chain composition and in terms of their C-C double bond position(s). Interestingly, hexadecenoic (16:1 Δ9) and octadecenoic (18:1 Δ9) along with octadecadienoic (18:2 Δ9,12) and octadecatrienoic (18:3 Δ9,12,15) were found to be the most abundant unsaturated fatty acyl chains of As-PL in the brown alga wakame, thus confirming it as a good source of essential fatty acids with a balanced ω6/ω3 ratio. Although the toxicity of As-including metabolites of algal As-PL is still a matter of debate and needs to be studied in more detail, the described approach can be exploited to assess if As-PL could contribute to the supply of essential fatty acids related to the use of algae as nutritious food.

Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides

We performed a systematic study of thirteen alkylamines used as ion-pairing reagents for ion-pair reversed-phase liquid chromatography (IP RP LC) separations of oligonucleotides on a C18 column. We proposed a method to classify the hydrophobicity of alkylamines by their retention in RP LC. The IP reagent hydrophobicity correlated with the retention and resolution of oligonucleotides in the corresponding IP mobile phases. The baseline resolution was achieved up to 30 mer for hydrophilic, or up to 50 mer for hydrophobic IP reagents. Hydrophobic alkylamines permitted useful oligonucleotide separations at relatively low buffer concentrations, such as 5–10 mM alkylamine-acetate IP systems. These buffers were compatible with mass spectrometry detection, however, replacement of acetic acid with hexafluoroisopropanol in the mobile phase improved the MS signal by 2–3 orders of magnitude. Experiments with native and chemically modified oligonucleotides highlighted the mixed-mode nature of IP RP LC. When using hydrophobic IP reagents, the ionic retention mechanism of oligonucleotides is enhanced while hydrophobic retention is diminished.

Sensitive and Robust LC-MS/MS Analysis of Salivary Cortisol in Negative Mode.

Cortisol is one of the most important glucocorticoids involved in the regulation of human metabolism and physiological stress. Monitoring of levels of cortisol is of immense clinical benefit. In particular, salivary cortisol levels have been shown to correlate well with diurnal changes in cortisol levels in serum and have been used widely for monitoring of cortisol levels for diagnosis and prognosis purposes. We present a sensitive, fast, and robust quantitative liquid chromatography and tandem mass spectrometry (LC-MS/MS) assay for salivary cortisol in negative mode. This assay employs protein precipitation followed by reversed-phase liquid chromatographic separation, negative-mode electrospray ionization (ESI), and MS/MS detection. This assay has a total run time of 5.8minutes and a limit of quantification of 0.5ng/mL with a linear range up to 100ng/mL. No carryover was observed at 10μg/mL. This assay also incorporates the routine monitoring of prednisolone, a potential interferent to salivary cortisol.

Preparative capillary electrophoresis (CE) fractionation of protein digests improves protein and peptide identification in bottom-up proteomics.

Reversed-phase liquid chromatography (RPLC) is widely used to reduce sample complexity prior to mass spectrometry (MS) analysis in bottom-up proteomics. Improving peptide separation in complex samples enables lower-abundance proteins to be identified. Multidimensional separations that combine orthogonal separation modes improve protein and peptide identifications over RPLC alone. Here we report a preparative capillary electrophoresis (CE) fractionation method that combines CE and RPLC separations. Using this method, we demonstrate improved protein and peptide identification in a tryptic digest of E. coli cell lysate, with 132 ± 33% more protein identifications and 185 ± 65% more peptide identifications over non-fractionated samples. Fractionation enables detection of lower-abundance proteins in this complex sample. We demonstrate improved coverage of ovarian cancer biomarker MUC16 isolated from conditioned cell media, with 6.73% sequence coverage using CE fractionation compared to 2.74% coverage without preparative fractionation. This new method will allow researchers performing bottom-up proteomics to harness the advantages of CE separations while using widely available LC-MS/MS instrumentation.

Monolithic Materials-Based RPLC-MS for Proteoform Separation and Identification.

High-performance separation of proteoforms plays an important role in top-down proteomic ananlysis due to high complexity of the proteome. To this end, the functionalized ethylene-bridged hybrid monolithic materials have been developed for reversed-phase liquid chromatographic separation of proteoforms followed by online combination with high-resolution mass spectrometry (MS) for top-down proteomic analysis. Such monoliths have advantages of homogenously distributed functional groups in the framework, good chemical stability, and high permeability and, thus, show high resolution, good reproducibility, and low backpressure for proteoform separation. This chapter describes in detail the preparation of such monoliths and online combination with high-resolution MS for proteoform separation and identification.

A new strategy for the computer-assisted development of reversed-phase liquid chromatography separation methods of unknown sample mixtures

A new strategy for the computer-assisted methods development in the reversed-phase liquid chromatographic separations of unknown sample mixtures has been developed using the latent spectral information in chromatogram raw data files of appropriately designed experiments, rather than resorting to elemental information functions (e.g., the number of peaks in chromatograms or similar criteria). The strategy developed allows unification of the approach for samples of both known and unknown composition and, thus, provide a general strategy for computer-aided tools in the chromatography laboratory. The operation principle of this strategy departs from extracting the spectra of components in the mixture chromatograms by resorting to multivariate curve resolution-alternating least squares (MCR-ALS). This technique allows the estimation of the true spectra for the individual components except when they have identical spectra or are fully overlapped. Thus, a convenient experimental design will try to perform separations of the sample mixture having at least partial resolution of components in some runs. This will allow estimating the spectra of components and, then, assign these components to the peaks in each run chromatogram. In this way, a retention model can be built for each component so computerized optimization process can be developed to provide the chromatographer with the best possible separation programs. Following this approach, strategies for sample mixtures of known and unknown composition are only different in the need of an initial spectrum discovery process for unknown mixtures and therefore a real general approach for the computer-assisted LC methods development is now available for the first time.

Sample Preparation Strategies for Antibody-Free Quantitative Analysis of High Mobility Group Box 1 Protein.

Sickness behavior and fatigue are induced by cerebral mechanisms involving inflammatory cytokines. High mobility group box 1 (HMGB1) is an alarmin, and a potential key player in this process. Reliable quantification methods for total HMGB1 and its redox variants must be established in order to clearly understand how it functions. Current methods pose significant challenges due to interference from other plasma proteins and autoantibodies. We aimed to develop an antibody-free sample preparation method followed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) to measure HMGB1 in human plasma. Different methods were applied for the removal of interfering proteins and the enrichment of HMGB1 from spiked human plasma samples. A comparison of methods showed an overall low extraction recovery (<40%), probably due to the stickiness of HMGB1. Reversed-phase liquid chromatography separation of intact proteins in diluted plasma yielded the most promising results. The method produced an even higher degree of HMGB1 purification than that observed with immunoaffinity extraction. Detection sensitivity needs to be further improved for the measurement of HMGB1 in patient samples. Nevertheless, it has been demonstrated that a versatile and fully antibody-free sample preparation method is possible, which could be of great use in further investigations.