An overview of the state-of-the-art in LC enantiomer separation is presented. This tutorial review is mainly focused on mechanisms of chiral recognition and enantiomer distinction of popular chiral ...selectors and corresponding chiral stationary phases including discussions of thermodynamics, additivity principle of binding increments, site-selective thermodynamics, extrathermodynamic approaches, methods employed for the investigation of dominating intermolecular interactions and complex structures such as spectroscopic methods (IR, NMR), X-ray diffraction and computational methods. Modern chiral stationary phases are discussed with particular focus on those that are commercially available and broadly used. It is attempted to provide the reader with vivid images of molecular recognition mechanisms of selected chiral selector–selectand pairs on basis of solid-state X-ray crystal structures and simulated computer models, respectively. Such snapshot images illustrated in this communication unfortunately cannot account for the molecular dynamics of the real world, but are supposed to be helpful for the understanding. The exploding number of papers about applications of various chiral stationary phases in numerous fields of enantiomer separations is not covered systematically.
Phosphoinositides (PIPx) play central roles in membrane dynamics and signal transduction of key functions like cellular growth, proliferation, differentiation, migration, and adhesion. They are ...highly regulated through a network of distinct phosphatidylinositol phosphates consisting of seven groups and three regioisomers in two groups (PIP and PIP2), which arise from phosphorylation at 3′, 4′, and 5′ positions of the inositol ring. Numerous studies have revealed the importance of both fatty acyl chains, degree of phosphorylation, and phosphorylation positions under physiological and pathological states. However, a comprehensive analytical method that allows differentiation of all regioisomeric forms with different acyl side chains and degrees of phosphorylation is still lacking. Here, we present an integrated comprehensive workflow of PIPx analysis utilizing a chiral polysaccharide stationary phase coupled with electrospray ionization high-resolution mass spectrometry with a data independent acquisition technique using the SWATH technology. Correspondingly, a targeted data mining strategy in the untargeted comprehensively acquired MS and MS/MS data was developed. This powerful highly selective method gives a full picture of PIPx profiles in biological samples. Herein, we present for the first time the full PIPx profiles of NIST SRM1950 plasma, Pichia pastoris lipid extract, and HeLa cell extract, including profile changes upon treatment with potential PI3K inhibitor wortmannin. We also illustrate using this inhibitor that measurements of the PIPx profile averaged over the distinct regioisomers by analytical procedures, which cannot differentiate between the individual PIPx isomers, can easily lead to biased conclusions.
Gold nanoparticles (GNPs) are often used as colloidal carriers in numerous applications owing to their low-cost and size-controlled preparation as well as their straightforward surface ...functionalization with thiol containing molecules forming self-assembling monolayers (SAM). The quantification of the ligand density of such modified GNPs is technically challenging, yet of utmost importance for quality control in many applications. In this contribution, a new method for the determination of the surface coverage of GNPs with thiol containing ligands is proposed. It makes use of the measurement of the gold-to-sulfur (Au/S) ratio by inductively coupled plasma mass spectrometry (ICP–MS) and its dependence on the nanoparticle diameter. The simultaneous ICP–MS measurement of gold and sulfur was carefully validated and found to be a robust method with a relative standard uncertainty of lower than 10%. A major advantage of this method is the independence from sample preparation; for example, sample loss during the washing steps is not affecting the results. To demonstrate the utility of the straightforward method, GNPs of different diameters were synthesized and derivatized on the surface with bifunctional (lipophilic) ω-mercapto-alkanoic acids and (hydrophilic) mercapto-poly(ethylene glycol) (PEG) n -carboxylic acids, respectively, by self-assembling monolayer (SAM) formation. Thereby, a size-independent but ligand-chain length-dependent ligand density was found. The surface coverage increases from 4.3 to 6.3 molecules nm–2 with a decrease of ligand chain length from 3.52 to 0.68 nm. Furthermore, no significant difference between the surface coverage of hydrophilic and lipophilic ligands with approximately the same ligand length was found, indicating that sterical hindrance is of more importance than, for example, intermolecular strand interactions of Van der Waals forces as claimed in other studies.
•Stereoselective separation of underivatized and AQC-derivatized amino acids on ZWIX.•Gradient elution mode with identical mobile phases using HPLC-ESI-QTOF-MS.•u-13C15N-L-Amino acid metabolomics ...mixture introduced as a peak assignment standard.•1,3,5-Tri-tert-butyl-benzene applied as void volume marker for mixed-mode columns.•Separation of isobaric amino acids and S-alkylated cysteine and homocysteine.
Amino acids play an important role in cellular processes and are building blocks for peptides and proteins, which take part in regulatory processes within each organism. Hence a large variety of biotechnologically or synthetically produced therapeutic drugs are peptides and proteins. Due to the chiral nature of amino acids and the large variety of common, uncommon and newly synthesized amino acid type compounds, stereoselective separation tools combined with mass spectrometric detection are important in research as well as purity control of therapeutics in industry. Since structural isomers and epimers of common amino acids are isobaric to each other, stereoselective separation is key to their identification. For this purpose zwitterionic quinine and quinidine type chiral stationary phases Chiralpak ZWIX(+) and Chiralpak ZWIX(-) were investigated for their separation performance for underivatized and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) derivatized proteinogenic amino acids, uncommon amino acids and their isobaric analogs such as allo-threonine, homoserine, allo-isoleucine and homocysteine by HPLC-ESI-QTOF-MS. Cystine and homocystine were reduced with dithiothreitol and S-alkylated with iodoacetic acid and iodoacetamide. In general, derivatization with AQC and thiol alkylation increased the detection sensitivity and resolution of acidic, basic and polar amino acids significantly (e.g. separation factor of Asp increased from 1.00 to 2.29 for Asp-AQC).
In addition, throughout this study a u-13C15N-L-amino acid metabolomics mixture was added to the DL-amino acid test solution and used as a co-eluting peak assignment standard to identify the corresponding u-12C14N-L-amino acid peak and hence determine the elution order of the enantiomer pairs for complex mixtures within a single run, employing the same separation conditions for underivatized and AQC-derivatized amino acids and their isobaric analogs.
Due to variation in instrument response caused by various sources of errors throughout an analytical assay, data normalization plays an indispensable role in untargeted LC-MS profiling, yet limited ...accepted guidelines on this topic exist. In this work, a systematic comparison of several normalization techniques, mainly focusing on internal standard-based approaches, has been performed to derive some general recommendations. For generation of untargeted lipidomic data, a comprehensive ultra-high performance liquid chromatography (UHPLC)-electrospray ionization (ESI)-quadrupole time of flight (QTOF)-MS/MS method was utilized. To monitor instrument stability and evaluate normalization performance, quality control (QC) samples, prepared from aliquots of all experimental samples, were embedded in the sequence. Stable isotope labeled standards, representing differing lipid classes, were spiked to each sample as internal standards for postacquisition normalization. Various metrics were used to compare distinct normalization strategies, with reduction of variation in QC samples being the critical requirement for acceptance of successful normalization. The comparison of intragroup coefficients of variation (CVs), median absolute deviations (MADs), and variance enables simple selection of the best performance of normalization with improved and coherent results. Furthermore, the importance for normalization in critical data sets, showing only minor effects between groups with high variation and outliers, is pointed out. Apart from normalization, also, influences of used raw data types are demonstrated. In addition, effects of various factors throughout the processing workflow were investigated and optimized. Eventually, implementation of quality control samples, even if not required for normalization, provided a useful basis for assessing data quality. Due to lack of consensus for selecting optimum normalization, suggestions for validating data integrity are given.
Inositol and inositol phosphates (IPx) are central metabolites. Their accurate quantitative analysis in complex biological samples is challenging due to lengthy sample preparation procedures, sample ...losses by strong adsorption to surfaces, and unpredictable matrix effects. Currently, U13C-inositol and U13C-IPx are not available from commercial sources. In this study, we developed a method that is capable of generating U13C-inositol and U13C-IPx. An inositol-independent cell line L929S was cultured in inositol-free medium supplemented with U13C-glucose. Inositol contamination in FBS was observed as the critical parameter for labeling efficiency (LE). A balance between cell growth and LE was achieved by adopting a two-step labeling strategy. In the first step, a LE of 90% could be obtained by normal cell growth in the long-term. Cells were then cultured in a second step in ultra-labeling medium for improved LE for a short duration before harvesting. The generated U13Canalogs were of high isotopic purity (>99%). Utilized as internal standards spiked before sample preparation in biological applications, U13Canalogs can effectively compensate sample loss during sample preparation as well as the matrix effect during electrospray ionization. An exemplary pharmacological study was conducted with phospholipase C inhibitor and activator to document the great utility of the prepared stable isotope-labeled internal standards in elucidating the PLC-dependent IP code. U13CIPx are used as internal standards to generate quantitative profiles of IPx in HeLa cell samples after treatment with PLC inhibitor and activator. This established method generating U13Canalogs is cost-effective, robust, and reproducible, which can facilitate quantitative studies of inositol and IPx in biological scenarios.
This work presents an advanced analytical platform for untargeted enantioselective amino acid analysis (eAAA) by comprehensive achiral × chiral 2D-LC hyphenated to ESI-QTOF-MS/MS utilizing ...data-independent SWATH (sequential window acquisition of all theoretical fragment-ion spectra) technology. The methodology involves N-terminal pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ) as retention, selectivity, and MS tag, supporting retention and UV detection in RPLC (1D), chiral recognition, and thus enantioselectivity by the core–shell tandem column composed of a quinine carbamate weak anion exchanger (QN-AX) and a zwitterionic chiral ion-exchanger (ZWIX(+)) (2D) as well as the ionization efficiency during positive electrospray ionization due to a high proton affinity of the AQC label. Furthermore, the urea-type MS tag gives rise to the generation of AQC-tag characteristic signature fragments in MS2. The latter allows the chemoselective mass spectrometric filtering of targeted and untargeted N-derivatized amino acids or related labeled species. The chiral core–shell tandem column provides a complete enantioselective amino acid profile of all proteinogenic amino acids within 1 min, with full baseline separation of all enantiomers, but without resolution of isomeric Ile/allo-Ile (aIle)/Leu, which can be resolved by RPLC. The entire LC × LC separation occurs within a total run time of 60 min (1D), with the chiral 2D operated in gradient elution mode and a cycle time of 60 s. A strategy to mine the 2D-LC-SWATH data is presented and demonstrated for the qualitative eAAA of two peptide hydrolysate samples of therapeutic peptides containing common and uncommon as well as primary and secondary amino acids. Absolute configuration assignment of amino acids using template matching for all proteinogenic amino acids was made feasible due to method robustness and the inclusion of an isotopically labeled L-U-13C15N-AA standard. The quantification performance of this LC × LC–MS/MS assay was also evaluated. Accuracies were acceptable for the majority of AAs enabling AA composition determination in peptide hydrolysates simultaneously with configuration assignment, as exemplified by oxytocin. This methodology represents a step toward truly untargeted 2D enantioselective amino acid analysis and metabolomics.
A simple, controllable, and reproducible stereoisomerization (racemization and epimerization) protocol for the preparation of scalemic α-amino acid mixtures from stereoisomerically pure standards was ...developed. Simply derivatize your amino acids with a racemization tag that incorporates a urea bond on the N-terminus of the target amino acid and incubate at elevated temperatures up to 95 °C for defined time periods until the targeted d-amino acid levels are obtained. The racemization tags investigated were 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC), aminophenyl-N-hydroxysuccinimidyl carbamate (AC), and 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APC). Employing this method, it was possible to create a ready-to-use, tailor-made chiral uniformly 13C and 15N labeled U- 13C15N-amino acid standard with the desired d-amino acid percentage within minutes or hours without sample cleanup. A racemization time of 30 min at 95 °C will lead to a d-amino acid level of 1–5%, while 6 h at 95 °C provides 15–30% d-amino acids. Racemization occurs due to imine formation at the chiral carbon atom bound to the urea-linking group without decomposition of labile amino acids such as Asn, Gln, Trp, Cit, and theanine. For amino acids possessing two chiral centers such as dl-Ile or dl-Thr, only the epimerization of isomers with different stereochemistry at the second chiral center will produce all four possible isobaric enantiomers. All measurements were performed on the zwitterionic Chiralpak ZWIX(+) column using a dual hydro-organic flow gradient combined with HPLC-ESI-QTOF-MS analysis. This new racemization method solves the problem of (enantioselective) matrix effects and inaccurate results in LC-MS based enantioselective metabolomics and warrants full MS-compatibility.
Fatty acyl-coenzyme As (acyl-CoAs) are of central importance in lipid metabolism pathways. Short-chain acyl-CoAs are usually part of metabolomics, and medium- to (very) long-chain acyl-CoAs are focus ...of lipidomics studies. However, owing to the specific complex and amphiphilic nature contributed by fatty acyl chains and hydrophilic CoA moiety, lipidomic analysis of acyl-CoAs is still challenging, especially in terms of sample preparation and chromatographic coverage. In this work, we propose a derivatization strategy of acyl-CoAs based on phosphate methylation. After derivatization, full coverage (from free CoA to C25:0-CoA) and good peak shape in liquid chromatography were achieved. At the same time, analyte loss due to the high affinity of phosphate groups to glass and metallic surfaces was resolved, which is beneficial for routine analysis in large-scale lipidomics studies. A sample preparation method based on mixed-mode SPE was developed to optimize extraction recoveries and allow optimal integration of the derivatization process in the analytical workflow. LC-MS/MS was performed with targeted data acquisition by SRM transitions, which were constructed based on similar fragmentation rules observed for all methylated acyl-CoAs. To achieve accurate quantification, uniformly 13C-labeled metabolite extract from yeast cells was taken as internal standards. Odd-chain and stable isotope-labeled acyl-CoAs were used as surrogate calibrants in the same matrix. LOQs were between 16.9 nM (short-chain acyl-CoAs) and 4.2 nM (very-long-chain acyl-CoAs). This method was validated in cultured cells and was applied in HeLa cells and human platelets of coronary artery disease patients. It revealed distinct acyl-CoA profiles in HeLa cells and platelets. The results showed that this method can effectively detect acyl-CoAs in biological samples. Considering their central importance in many de novo lipid biosynthesis and remodeling processes, this targeted method offers a valid foundation for future lipidomics analysis of acyl-CoA profiles in biological samples, particularly those concerning metabolic syndrome.
Fast liquid chromatography (LC) amino acid enantiomer separation of 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) derivatives using a chiral core–shell particle tandem column with weak anion ...exchange and zwitterionic-type quinine carbamate selectors in less than 3 min was achieved. Enantiomers of all AQC-derivatized proteinogenic amino acids and some isomeric ones (24 in total plus achiral glycine) were baseline separated (R s > 1.5 except for glutamic acid with R s = 1.3), while peaks of distinct amino acids and structural isomers (constitutional isomers and diastereomers of leucine and threonine) of the same configuration overlapped to various degrees. For this reason, drift tube ion mobility-mass spectrometry was added (i.e., LC-IM-MS) as an additional selectivity filter without extending run time. The IM separation dimension in combination with high-resolution demultiplexing enabled confirmation of threonine isomers (threonine, allo-threonine, homoserine), while leucine, isoleucine, and allo-isoleucine have almost identical collisional cross-section ( DTCCSN2 ) values and added no selectivity to the partial LC separation. Density functional theory (DFT) calculations show that IM separation of threonine isomers was possible due to conformational stabilization by hydrogen bond formation between the hydroxyl side chain and the urea group. Generally, the CCSN2 of protonated ions increased uniformly with addition of the AQC label, while outliers could be explained by consideration of intramolecular interactions and additional structural analysis. Preliminary validation of the enantioselective LC-IM-MS method for quantitative analysis showed compliance of accuracy and precision with common limits in bioanalytical methods, and applicability to a natural lipopeptide and a therapeutic synthetic peptide could be demonstrated.
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