Reversed-phase Liquid Chromatography Research Articles

Impact of fish oil supplementation on plasma levels of highly unsaturated fatty acid-containing lipid classes and molecular species in American football athletes

BackgroundPrevious studies have linked sports-related concussions and repeated subconcussive head impacts in contact sport athletes to elevated brain injury biomarkers. Docosahexaenoic acid (DHA), the primary omega-3 (n-3) highly unsaturated fatty acid (HUFA) in the brain, has shown neuroprotective effects in animal models after brain injury, but clinical research has shown mixed results.MethodsWe conducted a randomized, double-blind, placebo-controlled study on 29 Division 1 collegiate American football players, exploring the impact of DHA (2.5 g) and eicosapentaenoic acid (EPA) (1.0 g) supplied as ethyl esters, on levels of plasma lipids shown to cross the blood-brain barrier. Dietary intake data was collected using food frequency questionnaires (FFQ). Complex lipids and unesterified fatty acids were isolated from plasma, separated via reversed-phase liquid chromatography and analyzed by targeted lipidomics analysis.ResultsFFQ results indicated that participants had low dietary n-3 HUFA intake and high omega-6 (n-6):n-3 polyunsaturated fatty acids (PUFA) and HUFA ratios at baseline. After DHA + EPA supplementation, plasma lysophosphatidylcholine (LPC) containing DHA and EPA significantly increased at all timepoints (weeks 17, 21, and 26; p < 0.0001), surpassing placebo at Weeks 17 (p < 0.05) and 21 (p < 0.05). Phosphatidylcholine (PC) molecular species containing DHA or EPA, PC38:6 PC36:6, PC38:7, PC40:6, and PC40:8, increased significantly in the DHA + EPA treatment group at Weeks 17 (and 21. Plasma concentrations of non-esterified DHA and EPA rose post-supplementation in Weeks 17 and 21.ConclusionsThis study demonstrates that n-3 HUFA supplementation, in the form of ethyl esters, increased the DHA and EPA containing plasma lipid pools the have the capacity to enrich brain lipids and the potential to mitigate the effects of sports-related concussions and repeated subconcussive head impacts.Trial RegistrationAll deidentified data are available at ClinicalTrials.gov #NCT0479207.

Enhancing Sustainable Analytical Chemistry in Liquid Chromatography: Guideline for Transferring Classical High-Performance Liquid Chromatography and Ultra-High-Pressure Liquid Chromatography Methods into Greener, Bluer, and Whiter Methods.

This review is dedicated to sustainable practices in liquid chromatography. HPLC and UHPLC methods contribute significantly to routine analytical techniques. Therefore, the transfer of classical liquid chromatographic methods into sustainable ones is of utmost importance in moving toward sustainable development goals. Among other principles to render a liquid chromatographic method green, the substitution of the organic solvent component in the mobile phase with a greener one received great attention. This review concentrates on choosing the best alternative green organic solvent to replace the classical solvent in the mobile phase for easy, rapid transfer to a more sustainable normal phase or reversed-phase liquid chromatography. The main focus of this review will be on describing the transfer of non-green to green and white chromatographic methods in an effort to elevate sustainability best practices in analytical chemistry. The greenness properties and greenness ranking, in addition to the chromatographic suitability of seventeen organic solvents for liquid chromatography, are mentioned to have a clear insight into the issue of rapidly choosing the appropriate solvent to transfer a classical HPLC or UHPLC method into a more sustainable one. A simple guide is proposed for making the liquid chromatographic method more sustainable.

Addressing a major interference in the quantification of psilocin in mouse plasma: Development of a validated liquid chromatography tandem mass spectrometry method

Psilocybin is a psychedelic compound found in some hallucinogenic “magic mushrooms”. Psilocin is the active metabolite of Psilocybin, and it is the subject of several studies for the treatment of psychological disorders, such as anxiety, depression, and post-traumatic stress disorder. As such, the pharmacokinetic properties of psilocin should be evaluated to ensure its safety and efficacy as part of the drug development process. Based on the previously published studies, reversed-phase liquid chromatography (LC) was tested for psilocin quantification. The analysis, however, showed a major interference in mouse plasma that was not, to the best of our knowledge, reported previously. We, therefore, aimed to identify and separate the interference, using various chromatographic columns, mobile phase conditions, and mass spectrometers (MS) instruments. Chromatographic separation was achieved on an ultra high performance liquid chromatography (UHPLC) system, and a quadrupole-linear ion trap equipped with an electrospray ionization (ESI) source was used in positive ion mode with multiple reaction monitoring (MRM). Several chromatographic conditions and column chemistries, including C-18 and Phenyl-hexyl were initially tested, and failed to separate the interference. Exact mass measurement and MS/MS analysis were used to determine the structure of the interfering compound, which was confirmed to be tryptophan. Using the identified structure of the interfering compound, a fast and reliable hydrophilic interaction liquid chromatography (HILIC)-MS/MS method was developed and validated, that was capable of separating psilocin from the interference while achieving a 0.5 ng/ml lower limit of quantification (LLOQ). The validated method was successfully applied to a pharmacokinetic study where psilocin was orally administered to C57BL/6 mouse subjects. Psilocin concentration in all the analyzed mouse plasma samples was successfully determined.

The role of the cation and anion in aqueous liquid chromatography with sodium dodecyl sulphate and imidazolium-based ionic liquids as mobile phase reagents

BackgroundIn reversed-phase liquid chromatography, solute retention is primarily influenced by interactions between a nonpolar stationary phase and a moderately polar hydro-organic mobile phase, based on the solute lipophilicity. However, challenges regarding retention and peak tailing can arise due to ionic interactions between positively charged analytes and free silanols present on silica-based stationary phases. To address these challenges, incorporating surfactants and ionic liquids (ILs) into the mobile phase offers an effective solution. These additives synergistically enhance chromatographic performance through electrostatic and lipophilic interactions, which enable fine-tuning of selectivity and improved separation efficiency. ResultsThis study explores the chromatographic behaviour of several basic compounds in aqueous mixtures containing the anionic surfactant sodium dodecyl sulphate (SDS), above its critical micellar concentration, combined with various 1-alkyl-3-methylimidazolium-based ionic liquids (ILs) featuring chloride, tetrafluoroborate, and hexafluorophosphate anions, all without the addition of organic solvents. Specifically, this research investigates the influence of different anion types within the ILs and considers the impact of the IL cations. Analysis of solute peak profiles reveals narrow and symmetrical peaks. By introducing tetrafluoroborate and hexafluorophosphate IL anions into a mobile phase that contains an anionic surfactant, the study sheds light on the interactions occurring within the chromatographic column. This enhanced understanding of the combined effects of surfactants and ILs contributes to refining chromatographic methodologies. SignificanceThis research highlights the importance of carefully selecting the appropriate IL when incorporating it into a micellar mobile phase alongside SDS. This combination results in practical retention times that surpass the performance achieved with either the surfactant or IL alone in the mobile phase. The study particularly emphasises the impact of the IL anion, especially in the absence of SDS and organic solvents. This unveils interactions that are otherwise obscured in micellar and hydro-organic media, providing new insights into chromatographic dynamics.

Applications of high performance liquid chromatography-mass spectrometry in proteomics

Proteomics profiling plays an important role in biomedical studies. Proteomics studies are much more complicated than genome research, mainly because of the complexity and diversity of proteomic samples. High performance liquid chromatography-mass spectrometry (HPLC-MS) is a fundamental tool in proteomics research owing to its high speed, resolution, and sensitivity. Proteomics research targets from the peptides and individual proteins to larger protein complexes, the molecular weight of which gradually increases, leading to sustained increases in structural and compositional complexity and alterations in molecular properties. Therefore, the selection of various separation strategies and stationary-phase parameters is crucial when dealing with the different targets in proteomics research for in-depth proteomics analysis. This article provides an overview of commonly used chromatographic-separation strategies in the laboratory, including reversed-phase liquid chromatography (RPLC), hydrophilic interaction liquid chromatography (HILIC), hydrophobic interaction chromatography (HIC), ion-exchange chromatography (IEC), and size-exclusion chromatography (SEC), as well as their applications and selectivity in the context of various biomacromolecules. At present, no single chromatographic or electrophoretic technology features the peak capacity required to resolve such complex mixtures into individual components. Multidimensional liquid chromatography (MDLC), which combines different orthogonal separation modes with MS, plays an important role in proteomics research. In the MDLC strategy, IEC, together with RPLC, remains the most widely used separation mode in proteomics analysis; other chromatographic methods are also frequently used for peptide/protein fractionation. MDLC technologies and their applications in a variety of proteomics analyses have undergone great development. Two strategies in MDLC separation systems are mainly used in proteomics profiling: the "bottom-up" approach and the "top-down" approach. The "shotgun" method is a typical "bottom-up" strategy that is based on the RPLC or MDLC separation of whole-protein-sample digests coupled with MS; it is an excellent technique for identifying a large number of proteins. "Top-down" analysis is based on the separation of intact proteins and provides their detailed molecular information; thus, this technique may be advantageous for analyzing the post-translational modifications (PTMs) of proteins. In this paper, the "bottom-up" "top-down" and protein-protein interaction (PPI) analyses of proteome samples are briefly reviewed. The diverse combinations of different chromatographic modes used to set up MDLC systems are described, and compatibility issues between mobile phases and analytes, between mobile phases and MS, and between mobile phases in different separation modes in multidimensional chromatography are analyzed. Novel developments in MDLC techniques, such as high-abundance protein depletion and chromatography arrays, are further discussed. In this review, the solutions proposed by researchers when encountering compatibility issues are emphasized. Moreover, the applications of HPLC-MS combined with various sample pretreatment methods in the study of exosomal and single-cell proteomics are examined. During exosome isolation, the combined use of ultracentrifugation and SEC can yield exosomes of higher purity. The use of SEC with ultra-large-pore-size packing materials (200 nm) enables the isolation of exosomal subgroups, and proteomics studies have revealed significant differences in protein composition and function between these subgroups. In the field of single-cell proteomics, researchers have addressed challenges related to reducing sample processing volumes, preventing sample loss, and avoiding contamination during sample preparation. Innovative methods and improvements, such as the utilization of capillaries for sample processing and microchips as platforms to minimize the contact area of the droplets, have been proposed. The integration of these techniques with HPLC-MS shows some progress. In summary, this article focuses on the recent advances in HPLC-MS technology for proteomics analysis and provides a comprehensive reference for future research in the field of proteomics.

Fast and reliable method for analysis of derivatized plasma amino acids by liquid chromatography-single quadrupole-mass spectrometry

The pool of free, genetically encoded AA in plasma plays an essential role not only as substrate for every protein synthesized in the body but also as signaling molecules that regulate a wide range of physiological processes. Here we present a method for the analysis of 19 of the 20 encoded AA (except Cys) in dairy cow plasma. Isolated plasma or standards for the 19 AA were gravimetrically mixed with an internal standard mix consisting of 13C isotopes of each AA. Plasma proteins were precipitated on acetonitrile and supernatants transferred to glass vials. For pre-column derivatization, plasma supernatants were buffered with sodium borate (pH 9.5 −10), and AA were derivatized with 9-fluorenylmethoxycarbonyl (Fmoc) chloride. Analytes were isolated by solid-phase extraction using a strong-anionic ion exchange column and dry eluates were reconstituted in mobile phase consisting of 70% water solution of ammonium formate and 30% acetonitrile. Amino acid derivatives were separated by reverse phase liquid chromatography over 17.5 min with a C18 column in which acetonitrile increased to 80% over the first 11 min of the method, before returning to initial levels. Electrospray ionization on negative mode was used for most AA, except Arg and Pro, for which positive mode yielded superior results. Single or double (only Lys) derivatives were measured by single quadrupole mass spectrometry. We hypothesized that pre-column Fmoc derivatization would yield optimal resolution for quantitative analysis of the 19 targeted AA and their respective 13C internal standards, with limits of quantitation beyond physiological ranges. All the 19 AA were detected with minimal background noise. An 11-point standard curve was developed for each AA. Limits of quantitation were beyond concentrations observed in plasma samples of lactating dairy cows, except for Gly that upper curve points had to be removed to maintain linearity, limiting quantitation to the upper range of physiological concentration. After removing the 4 highest concentrations from the Gly standard curve, coefficients of determination were greater than 0.999 for all the AA. Recovery of spiked AA from plasma samples ranged from 89.9% for Phe to 100.3% for Trp. Instrument repeatability averaged 0.91 and ranged from 0.33 for Val to 2.29 for Arg. Meanwhile, sample preparation method repeatability averaged 2.02 and ranged from 1.14 for Tyr to 3.34 for Arg. While robust methods have been developed, they depend on either availability of sophisticated instruments, mostly limited to core facilities (i.e., tandem MS methods), long and expensive chromatography without specific internal standards for each AA (i.e., HPLC-UV and HPLC-FLD), or unstable derivatization (GC-MS). Here we describe a method with high throughput, stable derivatization, high precision and recovery, and potentially more affordable than most existing methods. This method could help dairy nutritionists to consider plasma AA information for diet formulation strategies, potentially reducing feeding costs and N emissions.

Propanediamine modified pillar[5]arene: A novel stationary phase for the high selectivity separation of versatile analytes

The unique properties of pillar[5]arene, including hydrophobic cavities, π-π conjugated and easy modification, make it a promising candidate as stationary phase for HPLC. Herein, we fabricated a novel propanediamine modified pillar[5]arene bonded silica as the stationary phase (PDA-BP5S) for reversed-phase liquid chromatography (RPLC). Benefiting from the significant hydrophobicity, π-π conjugative, p-π effect, and hydrogen bonding, the PDA-BP5S packed column showed high separation performance of versatile analytes involving polycyclic aromatic hydrocarbons, alkyl benzenes, phenols, arylamine, phenylethane/styrene/ phenylacetylene, toluene/m-xylene/mesitylene, halobenzenes, benzenediol and nitrophenol isomers. Especially, the separation of halobenzenes appeared to be controlled by both the size of the halogen substituents and the strength of the noncovalent bonding interactions, which was further confirmed by molecular dynamics simulation. The satisfactory separation and repeatability revealed the promising prospects of amine-pillar[5]arene-based stationary phase for RPLC.

Improved hydrophobic subtraction model of reversed-phase liquid chromatography selectivity based on a large dataset with a focus on isomer selectivity

Reversed-phase (RP) liquid chromatography is an important tool for the characterization of materials and products in the pharmaceutical industry. Method development is still challenging in this application space, particularly when dealing with closely-related compounds. Models of chromatographic selectivity are useful for predicting which columns out of the hundreds that are available are likely to have very similar, or different, selectivity for the application at hand. The hydrophobic subtraction model (HSM1) has been widely employed for this purpose; the column database for this model currently stands at 750 columns. In previous work we explored a refinement of the original HSM1 (HSM2) and found that increasing the size of the dataset used to train the model dramatically reduced the number of gross errors in predictions of selectivity made using the model. In this paper we describe further work in this direction (HSM3), this time based on a much larger solute set (1014 solute/stationary phase combinations) containing selectivities for compounds covering a broader range of physicochemical properties compared to HSM1. The molecular weight range was doubled, and the range of the logarithm of the octanol/water partition coefficients was increased slightly. The number of active pharmaceutical ingredients and related synthetic intermediates and impurities was increased from four to 28, and ten pairs of closely related structures (e.g., geometric and cis-/trans- isomers) were included. The HSM3 model is based on retention measurements for 75 compounds using 13 RP stationary phases and a mobile phase of 40/60 acetonitrile/25 mM ammonium formate buffer at pH 3.2. This data-driven model produced predictions of ln α (chromatographic selectivity using ethylbenzene as the reference compound) with average absolute errors of approximately 0.033, which corresponds to errors in α of about 3 %. In some cases, the prediction of the trans-/cis- selectivities for positional and geometric isomers was relatively accurate, and the driving forces for the observed selectivity could be inferred by examination of the relative magnitudes of the terms in the HSM3 model. For some geometric isomer pairs the interactions mainly responsible for the observed selectivities could not be rationalized due to large uncertainties for particular terms in the model. This suggests that more work is needed in the future to explore other HSM-type models and continue expanding the training dataset in order to continue improving the predictive accuracy of these models. Additionally, we release with this paper a much larger data set (43,329 total retention measurements) at multiple mobile phase compositions, to enable other researchers to pursue their own lines of inquiry related to RP selectivity.

Large-scale purification of a deprotected macrocyclic peptide by supercritical fluid chromatography (SFC) integrated with liquid chromatography in discovery chemistry

A macrocyclic peptide A was successfully purified in large quantities (∼30 g) in >95 % purity by an integrated two-step orthogonal purification process combining supercritical fluid chromatography (SFC) with medium-pressure reverse-phase liquid chromatography (MP-RPLC). MP-RPLC was used to fractionate the crude peptide A, remove unwanted trifluoroacetic acid (TFA) originating from the peptide A cleavage off the resin, and convert the peptide A into ammonium acetate salt form, prior to the final purification by SFC. A co-solvent of methanol/acetonitrile containing ammonium acetate and water in CO2 was developed on a Waters BEH 2-Ethylpyridine column. The developed SFC method was readily scaled up onto a 5 cm diameter column to process multi-gram quantities of the MP-RPLC fraction to reach > 95 % purity with a throughput/productivity of 0.96 g/h. The incorporation of SFC with MP-RPLC has been demonstrated to have a broader application in other large-scale polypeptide purifications.

Fibrous Polyimide Stationary Phase for the Separation of Aromatic Compounds in Reversed-Phase Liquid Chromatography

Fibrous Polyimide Stationary Phase for the Separation of Aromatic Compounds in Reversed-Phase Liquid Chromatography

Reversed-Phase Medium-Pressure Liquid Chromatography Purification of Omega-3 Fatty Acid Ethyl Esters Using AQ-C18.

Omega-3 fatty acids are in high demand due to their efficacy in treating hypertriglyceridemia and preventing cardiovascular diseases. However, the growth of the industry is hampered by low purity and insufficient productivity. This study aims to develop an efficient RP-MPLC purification method for omega-3 fatty acid ethyl esters with high purity and capacity. The results indicate that the AQ-C18 featuring polar end-capped silanol groups outperformed C18 and others in retention time and impurity separation. By injecting pure fish oil esters with a volume equivalent to a 1.25% bed volume on an AQ-C18 MPLC column using a binary isocratic methanol-water (90:10, v:v) mobile phase at 30 mL/min, optimal omega-3 fatty acid ethyl esters were obtained, with the notable purity of 90.34% and a recovery rate of 74.30%. The total content of EPA and DHA produced increased from 67.91% to 85.27%, meeting the acceptance criteria of no less than 84% set by the 2020 edition of the Pharmacopoeia of the People's Republic of China. In contrast, RP-MPLC significantly enhanced the production efficiency per unit output compared to RP-HPLC. This study demonstrates a pioneering approach to producing omega-3 fatty acid ethyl esters with high purity and of greater quantity using AQ-C18 RP-MPLC, showing this method's significant potential for use in industrial-scale manufacturing.

Green HPLC Enantioseparation of Chemopreventive Chiral Isothiocyanates Homologs on an Immobilized Chiral Stationary Phase Based on Amylose tris-[(S)-α-Methylbenzylcarbamate

Sulforaphane is a chiral phytochemical with chemopreventive properties. The presence of a stereogenic sulfur atom is responsible for the chirality of the natural isothiocyanate. The key role of sulfur chirality in biological activity is underscored by studies of the efficacy of individual enantiomers as chemoprotective agents. The predominant native (R) enantiomer is active, whereas the (S) antipode is inactive or has little or no biological activity. Here we provide an enantioselective high-performance liquid chromatography (HPLC) protocol for the direct and complete resolution of sulforaphane and its chiral natural homologs with different aliphatic chain lengths between the sulfinyl sulfur and isothiocyanate group, namely iberin, alyssin, and hesperin. The chromatographic separations were carried out on the immobilized-type CHIRALPAK IH-3 chiral stationary phase with amylose tris-[(S)-methylbenzylcarbamate] as a chiral selector. The effects of different mobile phases consisting of pure alcoholic solvents and hydroalcoholic mixtures on enantiomer retention and enantioselectivity were carefully investigated. Simple and environmentally friendly enantioselective conditions for the resolution of all chiral ITCs were found. In particular, pure ethanol and highly aqueous mobile phases gave excellent enantioseparations. The retention factors of the enantiomers were recorded as the water content in the aqueous-organic modifier (methanol, ethanol, or acetonitrile) mobile phases progressively varied. U-shaped retention maps were generated, indicating a dual and competitive hydrophilic interaction liquid chromatography (HILIC) and reversed-phase liquid chromatography retention mechanism on the CHIRALPAK IH-3 chiral stationary phase. Finally, experimental chiroptical studies performed in ethanol solution showed that the (R) enantiomers were eluted before the (S) counterpart under all eluent conditions investigated.

Two-dimensional sequential selective comprehensive chiral×reversed-phase liquid chromatography of synthetic phosphorothioate oligonucleotide diastereomers

In recent years, many nucleic acid-based pharmaceuticals have been approved and entered the market, and even a larger number are in late stage clinical trials. Conventional oligonucleotides are facing issues in vivo like fast renal clearance and nuclease degradation. Therefore, to increase their stability, phosphorothioation is a frequent modification of therapeutic oligonucleotides (ONs) which also leads to improved binding affinity facilitating cell internalization and intracellular distribution. At the same time, by replacing a phosphodiester linkage with a phosphorothioate group, a phosphorous stereogenic center is generated which causes the formation of Rp- and Sp-diastereomers. It increases the structural diversity. For example, with 15 of those phosphorothioate (PS) linkages, 32,768 different diastereomers are expected. Since the phosphorothioate is introduced non-stereoselectively, the molecular complexity of the resultant phosphorothioate ON products is tremendously increased impeding the chromatographic separation in the course of quality control. Since distinct phosphorothioate diastereomers have different bioactivities and pharmacological properties, there is increasing interest in implications of stereoisomerism of phosphorothiate oligonucleotides. From a quality and regulatory viewpoint, batch-to-batch reproducibility of the diastereomer profile may be of significant concern. In order to address this issue, this study investigates the stereoselectivity of LC methods for two phosphorothioate oligonucleotide (PSO) compounds differing in their molecular size and numbers of PS linkages. Diastereoselectivity of ion-pairing reversed-phase liquid chromatography (IP-RPLC), RPLC without ion-pairing agents and LC with chiral polysaccharide-based column were evaluated for model PSOs and an active pharmaceutical ingredient (API) of PSO with trivalent N-acetylgalactosamine (GalNAc) conjugate. Due to the structural complexity of PSOs, the separation power for the diastereomer mixture was increased by using sequential selective comprehensive two-dimensional chromatography with an amylose tris(α-methylbenzylcarbamate)-immobilized chiral stationary phase (CSP) in the first dimension and ion-pair RPLC with ethylammonium acetate in the second dimension. Improved diastereomer selectivity was obtained and a larger number of peaks could be separated.

Impact of ion-pairing systems choice on diastereomeric selectivity of phosphorothioated oligonucleotides in reversed-phase liquid chromatography

Ion-pairing reversed-phase liquid chromatography was utilized for the analysis of native and phosphorothioated oligonucleotides differing in the length (2–6mers and 21mer) and the number and position of phosphorothioate modifications. We investigated the influence of counterion (acetate vs. hexafluoroisopropanol) on the adsorption of eleven alkylamines on the stationary phases. A stronger adsorption of charged alkylamines on octadecyl- and phenyl-based stationary phases led to greater retention of oligonucleotides, and the adsorption of alkylamines was promoted with greater concentration of hexafluoroisopropanol in the mobile phase. Selected amines (triethylamine, dipropylamine, hexylamine) were used to study the resolution of n and n-x mers (main peak and its impurities shortened at 5´end), and diastereomeric separation of phosphorothioated oligonucleotides. The results confirmed a crucial role of alkylamine and counterion choice on the diastereomeric separation. The increasing hydrophobicity of alkylamine led to diminished diastereomeric selectivity which produced narrower phosphorothioated oligonucleotides peaks and led to improved n/n-x separation. Using hexafluoroisopropanol instead of acetate as counterion further enhances this effect (except for 100 mM concentration of hexafluoroisopropanol in combination with highly hydrophobic hexylamine). The elevated column temperature led to suppression of the diastereomeric resolution and improved resolution of n and n-x mers oligonucleotides. Baseline separation of oligonucleotides with different number of phosphorothioate linkages was achieved; this may be useful for therapeutic oligonucleotide analysis.

Ammonium bicarbonate buffers combined with hybrid surface technology columns improve the peak shape of strongly tailing lipids

BackgroundLipids such as phosphatidic acids (PAs) and cardiolipins (CLs) present strongly tailing peaks in reversed phase liquid chromatography, which entails low detectability. They are usually analyzed by hydrophilic interaction liquid chromatography (HILIC), which hampers high-throughput lipidomics. Thus, there is a great need for improved analytical methods in order to obtain a broader coverage of the lipidome in a single chromatographic method. We investigated the effect of ammonium bicarbonate (ABC) on peak asymmetry and detectability, in comparison with ammonium formate (AFO) on both a conventional BEH C18 column and an HST-CSH C18 column. ResultsThe combination of 2.5 mM ABC buffer pH 8 with an HST-CSH C18 column produced significantly improved results, reducing the asymmetry factor at 10 % peak height of PA 16:0/18:1 from 8.4 to 1.6. Furthermore, on average, there was up to a 54-fold enhancement in the peak height of its [M − H]- ion compared to AFO and the BEH C18 column. We confirmed this beneficial effect on other strongly tailing lipids, with accessible phosphate moieties e.g., cardiolipins, phosphatidylinositol phosphate, phosphatidylinositol bisphosphate, phosphorylated ceramide and phosphorylated sphingosine. Furthermore, we found an increased detectability of phospho- and sphingolipids up to 28 times in negative mode when using an HST-CSH C18 column. The method was successfully applied to mouse liver samples, where previously undetected endogenous phospholipids could be analyzed with improved chromatographic separation. SignificanceIn conclusion, the use of 2.5 mM ABC substantially improved the peak shape of PAs and enhanced the detectability of the lipidome in negative mode on an RPLC-ESI-Q-TOF-MS system on both BEH C18 and HST-CSH C18 columns. This method provides a wider coverage of the lipidome with one single injection for future lipidomic applications in negative mode.

Modelling the pH dependent retention and competitive adsorption of charged and ionizable solutes in mixed-mode and reversed-phase liquid chromatography

This study investigated the influence of pH on the retention of solutes using a mixed-mode column with carboxyl (-COOH) groups acting as weak cation exchanger bonded to the terminal of C18 ligands (C18-WCX column) and a traditional reversed-phase C18 column. First, a model based on electrostatic theory was derived and successfully used to predict the retention of charged solutes (charged, and ionizable) as a function of mobile phase pH on a C18-WCX column. While the Horváth model predicts the pH-dependent retention of ionizable solutes in reversed-phase liquid chromatography (RPLC) solely based on solute ionization, the developed model incorporates the concept of surface potential generated on the surface of the stationary phase and its variation with pH. To comprehensively understand the adsorption process, adsorption isotherms for these solutes were individually acquired on the C18-WCX and reversed-phase C18 columns. The adsorption isotherms followed the Langmuir model for the uncharged solute and the electrostatically modified Langmuir model for charged solutes. The elution profiles for the single components were calculated from these isotherms using the equilibrium dispersion column model and were found to be in close agreement with the experimental elution profiles. To enable modelling of two-component cases involving charged solute(s), a competitive adsorption isotherm model based on electrostatic theory was derived. This model was later successfully used to calculate the elution profiles of two components for scenarios involving (a) a C18 Column: two charged solutes, (b) a C18 Column: one charged and one uncharged solute, and (c) a C18-WCX Column: two charged solutes. The strong alignment between the experimental and calculated elution profiles in all three scenarios validated the developed competitive adsorption model.

A new chemical derivatization reagent sulfonyl piperazinyl for the quantification of fatty acids using LC-MS/MS

In our previous study, a chemical derivatization reagent named 5-(dimethylamino) naphthalene-1-sulfonyl piperazine (Dns-PP) was developed to enhance the chromatographic retention and the mass spectrometric response of free fatty acids (FFAs) in reversed-phase liquid chromatography coupled with electrospray ionization-mass spectrometry (RPLC-ESI-MS). However, Dns-PP exhibited strong preferences for long-chain FFAs, with limited improvement for short- or medium-chain FFAs. In this study, a new series of labeling reagents targeting FFAs were designed, synthesized, and evaluated. Among these reagents, Tmt-PP (N2, N2, N4, N4-tetramethyl-6-(4-(piperazin-1-ylsulfonyl) phenyl)-1,3,5-triazine-2,4-diamine) exhibited the best MS response and was selected for further evaluations. We compared Tmt-PP with Dns-PP and four commonly used carboxyl labeling reagents from existing studies, demonstrating the advantages of Tmt-PP. Further comparisons between Tmt-PP and Dns-PP in measuring FFAs from biological samples revealed that Tmt-PP labeling enhanced the MS response for about 80 % (30/38) of the measured FFAs, particularly for short- and medium-chain FFAs. Moreover, Tmt-PP labeling significantly improved the chromatographic retention of short-chain FFAs. To ensure accurate quantification, we developed a stable isotope-labeled Tmt-PP (i.e., d12-Tmt-PP) to react with chemical standards and serve as one-to-one internal standards (IS). The method was validated for accuracy, precision, sensitivity, linearity, stability, extraction efficiency, as well as matrix effect. Overall, this study introduced a new chemical derivatization reagent Tmt-PP (d12-Tmt-PP), providing a sensitive and accurate option for quantifying FFAs in biological samples.

On the Surprising Retention Order of Ketamine Analogs Using a Biphenyl Stationary Phase

An unexpected retention order for the ketamine analogs methoxpropamine (MXPr) and methoxisopropamine (MXiPr) was observed when using a biphenyl stationary phase for liquid chromatography-mass spectrometry (LC–MS). For separations of isomeric pairs of drugs (ketamine analogs, tryptamines, fentanyls, and nitazenes), branched-chain compounds are eluted before straight-chain compounds when using acetonitrile or methanol in mobile phases with C18 or biphenyl stationary phases. However, when a biphenyl stationary phase was paired with methanol, the opposite retention order for MXPr and MXiPr occurred, which was not observed when using acetonitrile in the mobile phase. The phenomenon persisted in biphenyl columns from two vendors. This study suggests additional work may be needed to understand the retention mechanisms when using biphenyl stationary phases. It is an example that we can still expect the unexpected when investigating alternative stationary phases of reversed-phase liquid chromatography (RPLC).

Tetrodotoxin Derivatization with a Newly Designed Boron Reagent Leads to Conventional Reversed-Phase Liquid Chromatography.

Tetrodotoxin (TTX) is a representative natural toxin causing pufferfish food poisoning, which is especially prominent in East and Southeast Asia, including Japan. TTX has been analyzed through post-column derivatization high-performance liquid chromatography (HPLC), ion-pair LC-MS(/MS), and hydrophilic interaction liquid chromatography (HILIC)-MS(/MS) as alternatives to the mouse bioassay method. However, post-column derivatization requires a system for online derivatization reactions, and with the ion-pair LC-MS approach, it is difficult to remove residual ion-pair reagents remaining in the equipment. Moreover, HILIC-MS provides poor separation compared to reversed-phase (RP) HPLC and requires a long time to reach equilibration. Therefore, we decided to develop a TTX analytical method using pre-column derivatization and RP HPLC for the rapid assessment of outbreak samples, including food remnants. In this study, we focused on the vic-diol moiety of TTX and designed a new derivatization reagent coded as NBD-H-DAB. This NBD-H-DAB was synthesized from 4-hydrazino-7-nitro-2,1,3-benzoxadiazole (NBD-H) and 3-fluoro-2-formylphenylboronic acid (FFPBA) with a simple reaction system and rapidly converted to its boronate form, coded NBD-H-PBA, in an aqueous reaction solution. The NBD-H-PBA demonstrated appropriate hydrophobicity to be retained on the RP analytical column and successfully detected with a UV spectrometer. It was easily reacted with the vic-diol moiety of TTX (C6 and C11) to synthesized a boronic ester. The derivatized TTX could be detected using the RP HPLC-UV, and the limit of detection in the fish flesh samples was 0.06 mg/kg. This novel pre-column derivatization of TTX with NBD-H-PBA proves capable for the analysis of TTX.

Hydrophilic Interaction Liquid Chromatography for the Analysis of Pharmaceutical Formulations

Abstract: For a long time, Reversed-Phase Liquid Chromatography (RPLC) was the most dominant technique for the analysis of pharmaceutical compounds, but with poor efficiency in the separation of small polar molecules. From the efforts to solve the problem of insufficient retention of these molecules, during the last decades, a mode of liquid chromatography named Hydrophilic Interaction Liquid Chromatography (HILIC) has experienced vast expansion. It is based on the use of a highly hydrophilic stationary phase along with an aqueous mobile phase with high organic modifier content. In this review, the characteristics of stationary and mobile phases used in HILIC are described, and corresponding separation mechanisms are discussed. An overview of recently published papers dealing with the application of HILIC in analyzing pharmaceuticals in biological and non-biological samples is provided. Besides, the application of HILIC systems in the determination of the physicochemical properties of compounds is described.