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Endocannabinoids are endogenous lipids with the main function recognized to act as neuromodulators through their cannabinoid receptors. Dysregulation of the endocannabinoid system is implicated in various pathologies, such as inflammatory and neurodegenerative diseases. In this study we describe a sensitive UHPLC-MS/MS method for the analysis of trace levels of 7 endocannabinoids in cerebrospinal fluid samples. The analytes covered comprised 1- and 2-arachidonoylglycerol 1- and 2-AG (which were analysed as sum due to their interconversion), 2-arachidonylglycerol ether 2-AGE, anandamide AEA, N-linoleoyl ethanolamide LEA, N-palmitoyl ethanolamide PEA and N-oleoyl ethanolamide OEA. Analytes were extracted from the biofluid by a simple monophasic procedure involving protein precipitation with acetonitrile (MeCN). The analytical method is based on chromatographic separation of the analytes with solid-core (core-shell, superficially porous) particle column Cortecs C18+ . Gradient elution with changing proportion of water and acetonitrile and constant concentration of formic acid provided reasonable separation of analytes, close elution of analytes and their internal standards and minimized matrix effects in biological samples. For specific detection of the endocannabinoids a triple-quadrupole tandem mass spectrometer with electrospray ionisation (ESI) and selected reaction monitoring (SRM) mode was used, and it provided good assay selectivity. The developed method required a minute volume of the biological samples (50 µL) and achieved excellent sensitivity (the lower limit of detection was between 4.15 and 30.18 pM of the biological sample). Linear calibration was achieved in the range from 25 to 10,545 pM for AEA, 90-3802 pM for 1-AG, 90-724 pM for 2-AG, 12-5226 pM for LEA, 33-13,942 for OEA, 34-23,850 pM for 2-AGE, 72-30,190 for PEA and 10-4218 for AEA-d4 in CSF. The method was validated and revealed relative errors in the range of - 14.7 to + 12.3% at LLOQ and - 14.1 to + 14.2% for the remaining validation range. Precisions were in the acceptable range (< 20% RSD at LLOQ, and

3-Hydroxyfatty acids (3-OH-FAs) are formed in the hydration step during mitochondrial β-oxidation of saturated straight-chain fatty acids, which is a catabolic pathway that involves several enzymes. For an unbiased biological interpretation, an enantioselective analysis of 3-OH-FAs including their stereoisomers is necessary, which may contribute to the elucidation of enzymatic mechanisms in the biological pathways. In this work, an enantioselective gradient UHPLC-MS/MS method based on 1.6 µm particle polysaccharide column (Chiralpak IA-U) for chiral separation of 3-hydroxyfatty acids was developed which covers carbon chain length from C8 to C18 with a good resolution of R and S enantiomers. The method is fast and sensitive for detecting enantiomers of 3-OH-FAs by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. A matrix matched-calibration strategy was applied for quantification of individual 3-OH-FA enantiomers. The method allows the simultaneous quantification of each enantiomer of 3-OH-FAs from C8-C18. One-phase liquid extraction with 2-propanol showed good extraction recoveries with over 90% on average. Further, the validated method was applied to investigate the alteration of 3-OH-FA enantiomers in platelets and plasma samples from human donors with different diagnoses of cardiovascular disease (acute coronary syndrome ACS, chronic coronary syndrome CCS). Both R and S enantiomers were detected in platelets and plasma samples with different predominance for R or S in dependence on carbon chain length, which might be associated with different functional enzymes of mitochondrial and peroxisomal β-oxidation. Finally, our study provides a new strategy for chiral separation and enantioselective analysis, showing great potential for targeted metabolomics in clinical biomarker discovery.

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U

Dental plaque is a structurally organized biofilm which consists of diverse microbial colonies and extracellular matrix. Its composition may change when pathogenic microorganisms become dominating. Therefore, dental biofilm or plaque has been frequently investigated in the context of oral health and disease. Furthermore, its potential as an alternative matrix for analytical purposes has also been recognized in other disciplines like archeology, food sciences, and forensics. Thus, a careful in-depth characterization of dental plaque is worthwhile. Most of the conducted studies focused on the screening of microbial populations in dental plaque. Their lipid membranes, on the other hand, may significantly impact substance (metabolite) exchange within microbial colonies as well as xenobiotics uptake and incorporation into teeth. Under this umbrella, a comprehensive lipidomic profiling for determination of lipid compositions of in vivo dental plaque samples and of in vitro cultivated biofilm as surrogate matrix to be used for analytical purposes has been performed in this work. An untargeted lipidomics workflow utilizing a ultra-high-performance liquid chromatography (UHPLC)-quadrupole-time-of-flight (QTOF) platform together with comprehensive SWATH (sequential window acquisition of all theoretical fragment ion mass spectra) acquisition and compatible software (MS-DIAL) that comprises a vast lipid library has been adopted to establish an extensive lipidomic fingerprint of dental plaque. The main lipid components in dental plaque were identified as triacylglycerols, followed by cholesterol, cholesteryl esters as well as diacylglycerols, and various phospholipid classes. In vivo plaque is a rare matrix which is usually available in very low amounts. When higher quantities for specific research assays are required, efficient ways to produce an appropriate surrogate matrix are mandatory. A potential surrogate matrix substituting dental plaque was prepared by cultivation of in vitro biofilm from saliva and similarities and differences in the lipidomics profile to in vivo plaque were mapped by statistical evaluation post-analysis. It was discovered that most lipid classes were highly elevated in the in vitro biofilm samples, in particular diacylglycerols, phosphatidylglycerols, and phosphatidylethanolamines (PEs). Furthermore, an overall shift from even-chain lipid species to odd-chain lipids was observed in the cultivated biofilms. On the other hand, even-chain phosphatidylcholines (PCs), lysoPCs, cholesteryl esters, and cholesterol-sulfate were shown to be specifically increased in plaque samples. Graphical abstract Electronic supplementary material The online version of this article (10.1007/s00216-019-02364-2) contains supplementary material, which is available to authorized users.

In this study, a combined targeted/untargeted UHPLC-ESI-QTOF-MS/MS method for the targeted quantitative analysis of the primary platelet lipid mediators thromboxane B2 (TXB2), 12S-hydroxy-5Z,8E,10E-heptadecatrienoic acid (HHT) and its oxidation product 12-keto-5Z,8E,10E-heptadecatrienoic acid (KHT) was developed, which allowed simultaneous untargeted profiling for the detection of other lipid biomarkers such as other oxylipins and fatty acids (FAs) in platelet releasates. A general procedure for the synthesis of keto-analogs from hydroxylated polyunsaturated FAs (PUFAs) using Dess-Martin periodinane oxidation reagent was proposed for the preparation of KHT standard. MS detection was performed in data independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) in the range of 50–500 Da with variable window sizes. The LC-MS/MS assay was validated for the targeted analytes and applied for analysis of supernatants derived from resting platelets and from platelets treated with thrombin. The targeted analytes KHT, HHT and TXB2 were found at highly elevated levels in the activated platelet releasates. On average, 13 ± 7, 15 ± 9, and 0.6 ± 0.2 attomols per platelet were released upon thrombin-activation. Furthermore, the simultaneous untargeted profiling (n = 8 in each group) revealed that these oxylipins are released with a pool of other (significantly upregulated) oxidized (12-HETE, 12-HEPE) and non-oxidized PUFAs. All these compounds can be considered additional biomarkers of platelet activation complementing the primary platelet activation marker thromboxane B2. The other lipids may support platelet activation or trigger other biological actions with some potential implications in thromboinflammation.

To find the best performing column for the analysis of protein-based biopharmaceuticals is a significant challenge as meanwhile numerous modern columns with distinct stationary phase morphologies are available for reversed-phase liquid chromatography. Especially when besides morphology also several other column factors are different, it is hard to decide about the best performing column a priori. To cope with this problem, in the present work 13 different reversed-phase columns dedicated for protein separations were systematically tested by the gradient kinetic plot method. A comprehensive comparison of columns with different morphologies (monolithic, fully porous and superficially porous particle columns), particle sizes and pore diameters as well as column length was performed. Specific consideration was also given to various monolithic columns which recently shifted a bit out of the prime focus in the scientific literature. The test proteins ranged from small proteins starting from 12 kDa, to medium sized proteins (antibody subunits obtained after IdeS-digestion and disulphide reduction) and an intact antibody. The small proteins cytochrome c, lysozyme and β-lactoglobulin could be analysed with similar performance by the best columns of all three column morphologies while for the antibody fragments specific fully porous and superficially porous particle columns were superior. A 450 Å 3,5 µm superficially porous particle column showed the best performance for the intact antibody while a 1.7 µm fully porous particle column with 300 Å showed equivalent performance to the best superficially porous column with thin shell and 400 Å pore size for proteins between 12 and 25 kDa. While the majority of the columns had C4 bonding chemistry, the silica monolith with C18 bonding and 300 Å mesopore size approximated the best performing particle columns and outperformed a C4 300 Å wide-pore monolith. The current work can support the preferred choice for the most suitable reversed-phase column for protein separations.

This work reports on targeted UHPLC-tandem mass spectrometry methods for the chiral separation of anteiso-methyl branched fatty acids (aiFAs). The methods involve precolumn derivatization with 1-naphthylamine and chiral separation on Chiralpak IG-U. anteiso-Methyl branched fatty acids with up to eight carbons can be separated. A method was used for the assignment of the absolute configuration of an aiFA present as fatty acyl residue of the teicoplanin mixture, namely teicoplanin RS3. Furthermore, the excellent methylene selectivity and improved selectivity for constitutional isomers of the polysaccharide columns was exploited for the elucidation and structural confirmation of previously unknown fatty acyl residues in teicoplanin. This shows the versatility and practical applicability of polysaccharide columns as orthogonal stationary phases to reversed-phase for structural elucidation of natural compounds. The developed methods are useful tools for related subdisciplines such as targeted metabolomics and lipidomics.

Enantioselective chromatography has been the preferred technique for the determination of enantiomeric excess across academia and industry. Although sequential multicolumn enantioselective supercritical fluid chromatography screenings are widespread, access to automated ultra-high-performance liquid chromatography (UHPLC) platforms using state-of-the-art small particle size chiral stationary phases (CSPs) is an underdeveloped area. Herein, we introduce a multicolumn UHPLC screening workflow capable of combining 14 columns (packed with sub-2 μm fully porous and sub-3 μm superficially porous particles) with nine mobile phase eluent choices. This automated setup operates under a vast selection of reversed-phase liquid chromatography, hydrophilic interaction liquid chromatography, polar-organic mode, and polar-ionic mode conditions with minimal manual intervention and high success rate. Examples of highly efficient enantioseparations are illustrated from the integration of chiral screening conditions and computer-assisted modeling. Furthermore, we describe the nuances of

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.

In this study, two monophasic isopropanol-water mixtures (IPA:H2O 75:25 v/v and IPA:H2O 90:10 v/v) were compared with traditionally employed biphasic methods of Bligh & Dyer and Matyash et al. as extraction systems for lipidomics analysis in Hela cells. Samples were analyzed by UHPLC-ESI-QTOF-MS/MS in positive and negative mode using sequential window acquisition of all theoretical fragment ion spectra (SWATH) and a relatively new software (MS-DIAL) was employed for the processing of the data which includes detection of peaks, MS/MS spectra deconvolution, identification of detected lipids and alignment of peaks through the analyzed samples. The studied performance parameters such as precision, recoveries of isotopically labeled internal standards and endogenous lipids, number of extracted lipids, and complexity of employed procedure showed that extraction with IPA:H2O 90:10 v/v performs similar to the Matyash protocol and better than Bligh & Dyer as well as IPA:H2O 75:25 v/v. However, less complex monophasic protocol which is simpler to implement and can be executed in plastic rather than glass, make the monophasic IPA:H2O 90:10 v/v protocol an excellent alternative to the classical biphasic protocols for reversed phase LC-MS lipidomics studies.

A non-targeted lipidomics workflow based on C8 core-shell particle ultra high-performance liquid chromatography (UHPLC) hyphenated to ESI-QTOF-MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion spectra (SWATH) was developed and applied to differential platelet lipidomics profiling of cardiovascular disease patients (stable angina pectoris (n = 10), ST-elevated myocardial infarction (n = 13)) against healthy controls (n = 10). DIA with SWATH generates comprehensive MS and MS/MS data throughout the entire chromatograms and all study samples. Hence, chromatograms can be extracted based on precursors or fragments which provided some benefits in terms of assay specificity in some cases. SWATH acquisition offers flexible experimental design with variable Q1 isolation windows. Liquid chromatography as well as SWATH settings were optimized to cover the lipidome of human platelets. The flexibility of the SWATH experiment design was utilized to implement target SWATH windows with narrow 5 Da Q1 precursor ion selection width (multiple reaction monitoring (MRM)-like SWATH windows) for the detection of low abundant oxidized phospholipids. Data processing was performed with MS-DIAL, and its feasibilities and caveats are discussed by illustrative examples. Thereby, identification of lipids is still a bottleneck in non-targeted lipidomics workflow. MS-DIAL, however, offers automatic identification via spectral matching using an in silico library. In total 1971 molecular features were detected cross the samples of which 611 were identified (total score >70%). The quality of the acquired data was validated with embedded quality control samples (n = 11). 80.3% of all features detected in the QC samples showed a coefficient of variation of below 30%. Multivariate statistics were used to visualize differences in the lipidome of distinct sample groups at a false discovery rate of 5%.

The present work systematically investigates the chemical microheterogeneity as part of the optimization of a single-step surface bonding chemistry of 3-mercaptopropylsilatrane (MPS) on mesoporous silica gel in comparison to the state-of-the-art silane chemistry with 3-mercaptopropyltrimethoxysilane (MPTMS). MPS functionalization turns out to be a favourable chemistry for the further use in thiol-ene click reactions such as the immobilization of chiral selectors, herein tert-butylcarbamoylquinine (tBuCQN), for the synthesis of chiral stationary phases (CSPs). MPS has higher reactivity than MPTMS and prefers the formation of trifunctional siloxane bondings unlike MPTMS which favours difunctional siloxane bonds to silica, as investigated by solid-state cross-polarization/magic angle spinning (CP/MAS) NMR (

In this work, we present a unique, robust and fully automated analytical platform technology for the enantioselective amino acid analysis using a multiple heart cutting RPLC-enantio/stereoselective HPLC-ESI-QTOF-MS method. This 2D-LC method allows the full enantioselective separation of 20 proteinogenic AAs plus 5 isobaric analogues, namely allo-Threonine (aThr), homoserine (Hse), allo-isoleucine (aIle), tert-Leucine (Tle) and Norleucine (Nle), after pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC; AccQ). This N-terminal AA-derivatization method introduces on the one hand beneficial chromatographic properties for 1D RP-LC (stronger retention) and 2D chiral separation (better chiral recognition), and on the other hand favorable detection properties with its chromophoric, fluorophoric, and easily ionizable quinoline mass tag. The entire separation occurs within a total 2DLC run time of 45 min, which includes the 1D-RP run and the 68 s 2D chiral separations of 30 heart-cuts (from the 1D-RP-run) on a chiral quinine carbamate (core-shell QNAX/fully porous ZWIX) tandem column. This relatively short overall run time was only possible by utilizing the highly efficient “smart peak parking” algorithm for the heart cuts and the resulting optimized analysis order thereof. 1D retention time precisions of This achiral-chiral 2DLC method was applied for the amino acid stereoconfiguration assignment of three peptides (aureobasidin A, a lipopeptide research sample, and octreotide) using an L-[u-13C15N] labelled internal AA standard mix spiked to each sample. The isotopically labelled L-AA standard allowed an easy and straightforward identification and configuration assignment, as well as the relative quantification of amino acids within the investigated peptides, allowing the direct determination of the number of respective amino acids and their chirality within a peptide.

Lipids play a major role in platelet signaling and activation. In this study, we analyzed the platelet lipidome in an untargeted manner by reversed-phase UHPLC for lipid species separation coupled to high-resolution QTOF-MS/MS in data-independent acquisition (DIA) mode with sequential window acquisition of all theoretical fragment ion mass spectra (SWATH) for compound detection. Lipid identification and peak picking was supported by the characteristic regular elution pattern of lipids differing in carbon and double bond numbers. It was primarily based on post-acquisition targeted feature extraction from the SWATH data. Multiple extracted ion chromatograms (EICs) from SWATH data of diagnostic ions on MS1 and MS2 level from both positive and negative ion mode allowed to distinguish between poorly resolved isomeric lipids based on their distinct fragment ions, which were used for relative quantification at a molecular lipid species level. It supports assay specificity for relative lipid quantitation via multiple quantifiably ions unlike to data-dependent acquisition methods which rely on precursor ions only. This approach was used to analyze human platelet samples. 457 lipids were annotated. Concentrations of lipids were estimated by stable isotope-labelled lipid class-specific internal standards as surrogate calibrants. Heatmaps of lipid concentrations in dependence on carbon and double bond numbers for the distinct lipid classes revealed a snapshot of the platelet lipidome in the resting state with lipid species distributions within classes supporting some functional interpretations. As expected, activation of the platelets by thrombin has led to significant alterations in the platelet lipidome as proven by univariate (volcano plot) and multivariate (PLS-DA) statistics. Several lipids were significantly up-regulated (lysophosphatidylinositols, oxylipins such as thromboxane B2 (TXB2), hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE), hydroxyoctadecadienoic acid (HODE), sphingoid-bases, (very) long chain saturated fatty acids) or down-regulated (lysophosphatidylethanolamines, polyunsaturated fatty acids, phosphatidylinositols). Several of them are well known as biomarkers of platelet activation while others may provide some further insights into pathways of platelet activation and platelet metabolism.

3-Hydroxy fatty acids are important chiral building blocks of lipopeptides and metabolic intermediates of fatty acid oxidation, respectively. The analysis of the stereochemistry of such biomolecules has significant practical impact to elucidate and assign the enzymatic specificity of the biosynthesis machinery. In this work, a new mass spectrometry compatible direct chiral ultra high performance liquid chromatography separation method for 3-hydroxy fatty acids without derivatization is presented. The application of amylose tris(3,5-dimethylphenyl carbamate) based polysaccharide chiral stationary phase immobilized on 1.6 μm silica particles (CHIRALPAK IA-U) allows the enantioseparation of 3-hydroxy fatty acids under generic electrospray ionization mass spectrometry friendly reversed phase gradient elution conditions. Adequate separation factors were achieved with both acetonitrile and methanol as organic modifiers, covering hydrocarbon chain lengths between C6 and C14 . Elution orders were derived from rhamnolipid (R-95) of which enantiomerically pure or enriched (R)-3-hydroxy fatty acids were recovered after ester hydrolysis. The S-configured acids consistently eluted before the respective R-enantiomers. The method was successfully applied for the elucidation of the absolute configuration of 3-hydroxy fatty acids originating from a novel lipopeptide with unknown structure. The work furthermore demonstrates that gradient elution is a viable option also in enantioselective (ultra)high performance liquid chromatography, even for analytes with modest separation factors, although less commonly exploited.

A short RNA with the sequence of the antisense strand of Patisiran has been selected as test material for the investigation of its common impurities using three different two-dimensional liquid chromatography (2D-LC) platforms. On the one hand, a quinine (QN) carbamate-based weak anion-exchange (AX) stationary phase (QN-AX) and a classical C18 reversed phase (RP) stationary phase in ion-pair (IP) mode with tripropylammonium acetate, respectively, have been used in the first dimension (1D) to provide the selectivity for impurities formed during the synthesis of the RNA. In the next step, certain peaks of interest from 1D have been transferred by multiple-heart-cutting (MHC) into a 2D in which an ESI-MS-compatible non-ionpairing RP method has been used for desalting via a diverter valve to remove non-volatile phosphate buffer components and ion-pair agents, respectively. Thus, a sensitive electrospray-ionization quadrupole time of flight mass spectrometry (ESI-TOF-MS) analysis of resolved impurity peaks of the siRNA has become possible under MS-friendly conditions. With both 2D-LC setups, peak purity of the ON has been evaluated by selective comprehensive (high resolution) sampling of the main peak. In a third MHC 2D-LC approach, the QN-AX LC mode was online coupled with the IP-RPLC in the 2D using UV detection. It allows the separation of additional impurities which coeluted in the first dimension. The potential of these methods for comprehensive impurity profiling of ON therapeutics is illustrated and discussed.

The profile of charge variants represents an important critical quality attribute of protein-based biopharmaceuticals, in particular of monoclonal antibodies, and must therefore becontrolled. In this work, 2D-LC methods for charge variant analysis were developed using a strong cation-exchange chromatography (SCX) as first dimension (1D) separation. Non-porous SCX (3 µm) particle columns and different mobile phases were evaluated using a test mixture of some standard proteins of different size and pI (comprising myoglobin, bovine serum albumin, cytochrome c, lysozyme and β-lactoglobulin) and two monoclonal IgG1 antibodies (NIST mAb and Secukinumab). The most promising 1D eluent for SCX was a salt-mediated pH-gradient system using a ternary mobile phase system with 2-(N-morpholino)ethanesulfonic acid, 1,3-diamino-2-propanol and sodium chloride. For the second dimension (2D), a desalting reversed-phase liquid chromatography (RP-LC) was chosen to enable the hyphenation of the charge variant separation with mass spectrometric (MS) detection. While for intact mAbs the 2D just served for desalting without additional selectivity, the 2D contributed some orthogonal selectivity for the mAb fragment separation. Various core-shell and monolithic columns were tested and variables such as gradient time and flow rate systematically optimized. Unexpectedly, a C4 400 A column (3.4 µm diameter with 0.2 µm porous shell) provided higher peak capacities compared to the same 1000 A column (2.7 µm diameter with 0.5 µm porous shell). A thinner shell appeared to be more advantageous than wider pores under high flow regime. An ultra-fast RP-LC method with a run time of one minute was developed using trifluoroacetic acid which was later replaced by formic acid as additive for better MS compatibility. The successful hyphenation of the two orthogonal separation modes, SCX and RP-LC, could be demonstrated in the multiple heart-cutting and the full comprehensive mode. MS analysis using a high-resolution quadrupole time-of-flight instrument enabled to identify different glycoforms and some major charge variants of the antibody at the intact protein level as well as on the subunit level (Fc/2, Lc, Fd') in a middle-up approach by 2D-LC-ESI-MS analysis.

There is a huge, still increasing market for synthetic and therapeutic peptides. Their quality control is commonly based on a generic reversed-phase liquid chromatography (RPLC) method with C18 stationary phase and acetonitrile gradient with 0.1% trifluoroacetic acid in the mobile phase. It performs exceptionally well for a wide variety of impurities, yet structurally closely related impurities with similar sequences, not resolved in preparative RPLC, may easily coelute in the corresponding QC run as well. To address this problem an advanced generic 2D-LC impurity profiling method was developed in this work. It employs a selective comprehensive (high resolution sampling) RP×RP 2D-LC separation using a 100×2.1 mm ID column with the common acidic generic gradient in the first dimension, while RPLC under basic pH on a short 30×3 mm ID column is used in the second dimension. Recording data with a UV detector at 215 nm after 1D separation provides the common generic 1D chromatogram. However, after the 2D separation a flow splitter enabled recording of the signals of complementary detectors comprising a diode array detector (DAD) in-line with a charged aerosol detector (CAD) and a quadrupole-time-of-flight (QTOF) mass spectrometer (MS) with an electrospray ionization (ESI) source. Generic conditions of this 2D-LC method have been established through optimization of 2D stationary and mobile phase considering different pH values and buffer concentrations. The orthogonal separation principle has been documented by a number of therapeutic peptides including Exenatide, Octreotide, Cyclosporine A and Oxytocin as well as some other proprietary synthetic peptides. The information density can be further enhanced by using the QTOF-MS detector by data independent acquisition with SWATH. Through this sequential window acquisition of all theoretical fragment ion mass spectra it became possible to collect MS/MS data comprehensively in the high-resolution sampling window, thus enabling the extraction of 2D-EICs from fragment ions and the generation of 2D-contour plots of all product ions. Using Oxytocin as an example for an important therapeutic peptide, the ability of this advanced generic sRP-UV×RP-DAD-CAD-ESI-QTOF-MS/MS method with SWATH for peptide quality control is discussed.

Oxylipins, the oxidation products of polyunsaturated fatty acids, are important signaling molecules in living organisms. Some of them have pro-inflammatory properties, while others act as pro-resolving agents. Oxylipins also play a major role in platelet biology and the progression of thrombo-inflammation. Depending on their structure, they may be pro-thrombotic or anti-thrombotic. For an unbiased biological interpretation, a detailed analysis of a broad spectrum of oxylipins including their stereoisomers is necessary. In our work, we developed for the first time an enantioselective UHPLC-ESI-MS/MS assay which allows quantifying individual oxylipin enantiomers. The assay made use of a sub-2µm particle-based amylose-(3,5-dimethylphenylcarbamate) chiral stationary phase (Chiralpak IA-U) under MS-compatible reversed-phase conditions. It covered 19 enantiomeric pairs of oxylipins and one diasteromeric pair of a lipid mediator: 2 pairs of hydroxyoctadecadienoic acids (HODE), 6 pairs of hydroxyeicosatetraenoic acids (HETE), 5 pairs of hydroxyeicosapentaenoic acids (HEPE), 3 pairs of hydroxydocosahexaenoic acids (HDoHE) and one pair of each: resolvins D1, hydroxyeicosatrienoic acid (HETrE), hydroxyoctadecatrienoic acid (HOTrE) and dihydroxyeicosatetraenoic acid (DiHETE). The new method is fast and showed outstanding peak resolution for most of the isomeric pairs. Excellent method sensitivity (average LOD was equal to 2.7 pg on column) was obtained by using a triple quadrupole instrument as a detector in a targeted, selected reaction monitoring (SRM) mode. The applicability of the method was verified by preliminary validation. It was then applied to analyze oxylipins produced by autoxidation of polyunsaturated fatty acids (PUFA) in air. Multiple oxylipins were found in each of the samples as racemic mixtures and served as reference substances for identification. Finally, the new enantioselective UHPLC method was applied to analyze releasates from platelets in resting state, and following activation with thrombin. The highest abundant oxylipin in the platelet releasate was 12(S)-HETE, but many other oxylipins were found in the thrombin activated samples, usually as single enantiomers (e.g. 12(S)-HEPE, 11(R)-HETE, 9(R)-HODE, 13-(S)-HODE, 14(S)-HDoHE). The latter was detected at about similar concentration in resting platelet releasates as well. 15-HETE showed elevated levels for both R-and S-enantiomers in releasates of thrombin-activated platelets. 12-HETrE was found presumably as both enantiomers, however, retention time inconsistencies indicate that the R-enantiomer is actually a different compound, maybe another constitutional isomer with different double-bond configuration.

Undaria pinnatifida (Wakame) alga contains high amounts of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid which was reported to decrease the efficiency of cisplatin chemotherapeutics. To obtain a fatty acid enriched extract of this ω-3 poly-unsaturated fatty acid as an analytical standard, Wakame was used as source material for its extraction. A two-step extraction protocol consisting of a liquid-liquid extraction followed by solid-phase extraction with 3-aminopropyl silica in accordance to a normal-phase elution mode was developed. An ultra high performance liquid chromatography with electrospray ionization tandem mass spectrometry method based on sequential windowed acquisition of all theoretical fragment ion mass spectra allowed a simultaneous comprehensive group selective fatty acids profiling in untargeted manner and quantitative analysis of the targeted fatty acid. Hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid was identified using high-resolution product ion spectra. The quantitative method was based on d5-deuterated hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid which was employed as surrogate calibrant. Preliminary method validation was performed by evaluating detection and quantification limits, linear range, intra-assay and inter-day precision. Finally, a concentration of 421.2 ± 14.9 ng/mL (4% CV) of hexadeca-4Z,7Z,10Z,13Z-tetraenoic acid was determined in the extract which was further used as analytical standard.

Recent advancements in particle design are common in reversed-phase liquid chromatography (RPLC), but in chiral separations their use is still sporadic in commercially available chiral stationary phases (CSPs). Due to reported lower mass transfer resistance, they might be a promising opportunity to increase efficiency and reduce time of analysis since the relatively higher mass transfer resistance term of CSPs caused by slow adsorption-desorption kinetics is the most performance-limiting factor in enantioselective chromatography. This study was dedicated to the evaluation of new support materials for tert-butylcarbamoylquinine (tBuCQN) based CSP to provide highly efficient and fast enantioseparations. As the main focus of this study, the chiral selector tBuCQN was immobilized on sub-2 μm fully porous particles (FPPs) and 2.7 μm superficially porous particles (SPPs) and their column performance in enantioseparation was evaluated in comparison to 5 μm FPPs by van Deemter and Knox analyses as well as kinetic plots using racemic Fmoc-Phe. Both new particle types outperformed the 5 μm FPP benchmark in terms of speed and efficiency, with wider pore materials (160 or 200 A) being advantageous (over 90 or 120 A). Basically decisive for the performance gain was the 10-times smaller mass transfer resistance. Furthermore, 2.7 μm 160 A SPPs outperformed their fully porous sub-2 μm 120 A counterpart (HminR = 4.64 μm vs. HminR = 8.94 μm) due to various parameters affording reduced plate height h of 1.7. Caused by the inaccessible core, separations were about 2-times faster. Packing of 2.7 μm core-shell particles provided a very homogeneous column bed, and, owing to its higher permeability, the column backpressure was much lower. It enables packing of longer columns providing theoretically separation efficiencies of up to 106 plates per m (as indicated by kinetic plots) and versatile use without the necessity of UHPLC systems. Investigating the effect of particle size reduction (FPPs: 5 μm, 3 μm, 1.7 μm; SPPs: 2.7 μm, 2 μm) and wider pores (FPPs: 120 A, 200 A; SPPs: 90 A, 160 A), a significantly reduced mass transfer resistance was the driving force for performance gain. Individual contributions of peak dispersion were deconvoluted for 5 μm FPP CSP and confirmed that slow adsorption-desorption kinetics is the most significant contribution to peak broadening in this chromatographic system.

A multiple heartcut (MHC) 2D-UHPLC method with UV detection has been developed for the enantioselective analysis of complex amino acid mixtures in a single run. The MHC method is based on an achiral gradient RPLC separation with 1.8 μm C18 phase (100 × 2.1 mm ID column) in the first dimension (1D) and enantioselective isocratic separation on a tert-butylcarbamoylquinine-based 2.7 μm Coreshell particle column (50 × 3 mm ID) in the second dimension (2D). Pre-column derivatization has been performed with Sanger’s reagent (2,4-dinitrofluorobenzene) yielding chromogenic 2,4-dinitrophenylated amino acids (DNP-AAs). Heartcuts of 40 μL fractions of the 1D peaks were sampled into the 2D system via a two-position four-port dual valve connected to two loop decks each equipped with six 40 μL parking loops. Using this setup, 25 amino acids (20 proteinogenic plus allo-Thr, allo-Ile, homoserine (Hse), Orn, β-Ala) have been analyzed enantioselectively in a fully automated manner with a single chiral column within 130 min total run time (1D and 2D). All 2D separations together took 101.5 min (29 cuts with 3.5 min run time each) and thus the total analysis time was quite efficiently utilized. Faster separations were restricted by some software constraints which did not allow to adjust run times in 2D individually. The practical utility of this enantioselective MHC method is documented by application for the absolute configuration determination of the amino acids in gramicidin and bacitracin. Further optimizations should lead to a generic enantioselective amino acid analyzer for the quality control of synthetic peptides and the structural characterization of non-ribosomal peptides.

Quality-by-Design approach for enantioselective HPLC method development surpasses Quality-by-Testing in offering the optimal separation conditions with the least number of experiments and in its ability to describe the method's Design Space visually which helps to determine enantiorecognition to a significant extent. Although some schemes exist for enantiomeric separations on Cinchona-based zwitterionic stationary phases, the exact design space and the weights by which each of the chromatographic parameters influences the separation have not yet been statistically studied. In the current work, a screening design followed by a Response Surface Methodology optimization design were adopted for enantioseparation optimization of 3 model drugs namely the acidic Fmoc leucine, the amphoteric tryptophan and the basic salbutamol. The screening design proved that the acid/base additives are of utmost importance for the 3 chiral drugs, and that among 3 different pairs of acids and bases, acetic acid and diethylamine is the couple able to provide acceptable resolution at variable conditions. Visualization of the response surface of the retention factor, separation factor and resolution helped describe accurately the magnitude by which each chromatographic factor (% MeOH, concentration and ratio of acid base modifiers) affects the separation while interacting with other parameters. The global optima compromising highest enantioresolution with the least run time for the 3 chiral model drugs varied extremely, where it was best to set low % methanol with equal ratio of acid-base modifiers for the acidic drug, very high % methanol and 10-fold higher concentration of the acid for the amphoteric drug while 20 folds of the base modifier with moderate %methanol were needed for the basic drug. Considering the selected drugs as models for many series of structurally related compounds, the design space defined and the optimum conditions computed are the key for method development on cinchona-based chiral stationary phases.

Cortisol and cortisone are common markers for stress and thus preferentially analyzed in matrices that allow non-invasive sampling such as saliva. Though the major drawback of immunoassays is lack of specificity due to cross reactivities, they are still most commonly used for quantification of steroid hormones. To overcome such problems, sensitive methods based on liquid chromatography-mass spectrometry are becoming more and more accepted as the golden standard for steroid bioanalysis as they achieve accurate quantification at trace levels for multiple analytes in the same run. Along this line, the aim of this study was the development of a new microflow UHPLC-ESI-MS/MS method for the measurement of salivary cortisol and cortisone, which due to its microflow regime provides enhanced sensitivity and is more ecofriendly. The developed method implemented sample preparation by Solid-Phase Extraction (SPE) in a 96-well plate format. Data acquisitions were carried out in MRM (multiple reaction monitoring) mode. The quantitative determination of endogenous compounds in saliva remains a challenge since analyte-free matrix is lacking. Hence, a surrogate calibrant approach with cortisol-d4 and cortisone-13C3 was applied for the target compounds in the presented method. A number of factors were optimized and the method validated. The lower limit of quantitation (LLOQ) was 72 and 62 pg mL−1 for cortisol and cortisone, respectively. Linear calibration was achieved in the range from 0.062 to 75.5 ng mL−1 for cortisol-d4 and 0.072 to 44 ng mL−1 for cortisone-13C3. The performance of the method was also evaluated via proficiency test for salivary cortisol. Finally, it was applied successfully to evaluate cortisol and cortisone concentrations in multiple batches in routine clinical stress study samples (4056 total injections with 1983 study samples). Moreover, the instrument performance (in particular retention time variability) within each batch, between different batches and lot-to-lot of 5 investigated capillary columns over time is described. The work documents that micro-UHPLC-ESI-MS/MS is suitable and robust enough to carry out a full clinical study with greater than 1000s of samples over an extended period if adequate internal standards can be used.

Chiral 2-hydroxycarboxylic acids are compounds that have been linked to particular diseases and are putative biomarkers with some diagnostic potential. The importance of identifying whether a particular enantiomer is related to certain diseases has been encouraged recently. However, in many cases it has not yet been elucidated whether there are stereochemical implications with respect to these biomarkers and whether their enantioselective analysis provides new insights and diagnostic potential. In this study 13 disease-related chiral 2-hydrocarboxylic acids were studied for their chiral separation by high-performance liquid chromatography on three cinchona alkaloid-derived chiral stationary phases. From a subgroup of eight 2-hydroxymonocarboxylic acids, baseline resolution could be achieved and inversion of elution order by exchanging tert-butylcarbamoyl quinidine chiral stationary phase (Chiralpak QD-AX) for the corresponding quinine analogue (Chiralpak QN-AX) is shown for seven of them. Furthermore, conditions for chiral separation of the 2-hydroxydicarboxylic acids, citramalic acid, 2-isopropylmalic acid, and 2-hydroxyadipic acid are reported and compared to the previous reported conditions for 2-hydroxyglutaric acid and malic acid.

Chiral short chain aliphatic hydrocarboxylic acids (HCAs) are common compounds being part of different biological processes. In order to control and understand these processes is of pivotal importance to determine the identity of the involved enantiomer or their enantiomeric ratio. In this study the capacity of quinine- and quinidine-derived chiral stationary phases to perform the enantioseparation of eight chiral HCAs (tartaric acid, isocitric acid, malic acid, glyceric acid, 2-hydroxyglutaric acid, 2-hydroxybutyric acid, lactic acid and 3-hydroxybutyric acid) was evaluated. MS-compatible conditions consisting of ACN/MeOH mixtures as eluents with formic acid, acetic acid and/or their ammonium salts as additives, temperatures between 10 and 25 °C (except for −20 °C for 3-hydroxybutyric acid) and a flow rate of 1.00 mL/min yielded full baseline resolution for all studied HCAs. Elution order for the HCA enantiomers was determined revealing different behaviors between the studied compounds.

The successful fabrication of monolithic capillary columns for enantiomer separations was achieved within vinylized fused silica capillaries via fast "one-pot" photo-initiated free radical polymerization reaction. A mixture consisting of polyhedral oligomeric silsesquioxane, O-[2-(methacryloyloxy)ethylcarbamoyl]-10,11-dihydroquinidine was copolymerized in the presence of n-butanol, ethylene glycol and photo-initiator 2,2-dimethoxy-2-phenylacetophenone. The morphology of the resultant polymeric hybrid inorganic-organic material and its permeability as well as porosity can be controlled by adjusting the composition of the monomers and binary porogenic solvent. The chromatographic characteristics of the columns have been investigated. Separation factors of N-acetyl-phenylalanine (Ac-Phe) and dichlorprop dropped with decrease of chiral functional monomer. Permeability was better when the macroporogen ethyleneglycol was present at higher concentrations during the polymerization. In general, the chiral compounds were well separated (dichlorprop: α = 1.53, Rs up to 4.14; Ac-Phe: α = 1.36, Rs up to 2.69) by nano-HPLC with an optimized enantioselective monolithic capillary column which can be prepared within a few minutes.

The aim of this research study was to provide a more thorough understanding of the underlying mechanism and to broaden the application field of the recently introduced racemization method employing the amino acid derivatization tag 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC, AccQ) for heat-induced stereoisomerization of common amino acids as well as uniformly isotopically labeled [U-13C15N]-amino acids. The influence of different buffer types such as sodium borate buffer and sodium carbonate buffer as well as their pH and molarity on the racemization and deamidation of amino acids were investigated. It was found that a 0.4 M borate buffer with a pH of 8.0 +/− 0.2 was the most suitable derivatization as well as racemization buffer to ensure degradation free racemization of deamidation prone compounds such as glutamine. Hereby essential was the in-solution pH measurement before and after derivatization with AQC as well as after heat-induced racemization. This strategy provided further insight at which pH an actual racemization event was observed and when an unwanted deamidation of glutamine to glutamic acid occurred. In addition also the influence of the presence of oxygen during racemization was studied. In this context it was possible to determine ideal oxidation and racemization conditions for the production of scalemic mixtures of chiral isotopically labeled methionine AQC-DL-[U-13C15N]-Met as well as its oxidation products, AQC-DL-[U-13C15N]-Met-O and AQC-DL-[U-13C15N]-Met-O2. All stereoselective separations were performed on the zwitterionic Chiralpak ZWIX(+) column combined with HPLC–ESI–QTOF–MS analysis in positive ionization mode.

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.

Lipidomics has gained rising attention in recent years. Several strategies for lipidomic profiling have been developed, with targeted analysis of selected lipid species, typically utilized for lipid quantification by low-resolution triple quadrupole MS/MS, and untargeted analysis by high-resolution MS instruments, focusing on hypothesis generation for prognostic, diagnostic and/or disease-relevant biomarker discovery. The latter methodologies generally yield relative quantification data with limited inter-assay comparability. In this work we aimed to combine untargeted analysis and absolute quantification to enhance data quality and to obtain independent results for optimum comparability to previous studies or database entries. For the lipidomic analysis of mouse plasma, RP-UHPLC hyphenated to a high-resolution quadrupole TOF mass spectrometer in comprehensive data-independent SWATH acquisition mode was employed. This way, quantifiable data on the MS and the MS/MS level were recorded, which increases assay specificity and quantitative performance. Due to the lack of an appropriate blank matrix for untargeted lipidomics, we herein established a sophisticated strategy for lipid class-specific calibration with stable isotope labeled standards (surrogate calibrants). LLOQs were in the range between 10 and 50 ng mL−1 for LPC, LPE, PI, PS, PG, SM, PC, PE, DAG) or 100–700 ng mL−1 (MAG, TAG), except for cholesterol and CE (1–20 μg mL−1). Acceptable values for accuracy and precision well below ±15% bias were reached for the majority of surrogate calibrants. However, to achieve sufficient accuracy for target lipids, response factors to corresponding surrogate calibrants are required. An approach to estimate response factors via a standard reference material (NIST SRM 1950) was therefore conducted. Furthermore, a useful workflow for post-acquisition re-calibration, involving response factor determination and iteratively built libraries, is suggested. In comparison to single-point calibration, the presented surrogate calibrant method was shown to yield results with improved accuracy that are largely in accordance with standard addition. Quantitative results of real samples (high-fat diet vs control diet) were then compared to two previously published dietary mouse plasma studies that provided absolute lipid levels and showed similar trends.

Betulin is a pentacyclic triterpene with demonstrated healing properties in mid-dermal wounds. A few earlier studies have provided insights into the wound healing effects on the molecular level. However, there are still questions left on the molecular targets of betulin. Therefore, a pharmacolipidomics analysis of betulin is undertaken in human immortalized keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. For this purpose, lipid extracts of keratinocytes treated with betulin and untreated controls are comprehensively analyzed by an untargeted UHPLC-ESI-QTOF-MS/MS lipidomics profiling workflow using data-independent acquisition. Targeted data processing allows the identification of 611 lipid species from 21 different lipid classes. Statistical analysis of the identified lipids shows significant changes in 440 lipid species that can be described as downregulation of cholesteryl esters and triacylglycerides and upregulation of glycerophospholipids, sphingolipids, and diacylglycerides. Additionally, some other signals corresponding to triterpenes are found in the betulin group and suggested that betulin is incorporated (in the membrane) and metabolized in keratinocytes.

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.

Zwitterionic chiral ion-exchange selectors (ZWIX) obtained by conjugation of quinine and 2-aminocyclohexanesulfonic acid via a carbamate bond were immobilized on three different silica particle types, viz. 120 A 3 μm fully porous particles (FPP), 200 A 3 μm FPP and 160 A 2.7 μm superficially porous particles (SPP). Selector densities were determined by elemental analysis and the porosities of packed columns measured by inverse size exclusion chromatography with polystyrene standards. Liquid chromatographic tests with a set of chiral zwitterionic, acidic and basic analytes showed that the surface chemistry was successfully transferred to the distinct particle morphologies. The chromatographic performance of the three columns was evaluated by acquiring van Deemter curves. The results showed that the column packed with the SPP particles gives the best performance and kinetic plots further demonstrated that they represent the most favorable compromise in terms of speed, efficiency and pressure drop. Sub-minute separations could be accomplished at much lower pressure drop on the core-shell column, e.g. 2-amino-2-phenylbutyric acid was baseline separated in less than 15 s on a 5 cm long column. The Maxwell effective medium theory with second order approximation was applied to calculate effective diffusion in the mesoporous zones of SPP and FPP, which allowed eventually to deconvolute the individual peak dispersion contributions (ha, hb, hc,m, hc,s, hc,ads). The efficiency gain of the 160 A SPP column compared to the 120 A FPP (benchmark) column was mainly due to lower eddies (ha), smaller c-term accounting for slow adsorption-desorption kinetics in enantioselective chromatography (hc,ads), and also due to lower stationary mass transfer resistance (hc,s). Enhanced effective diffusion (Deff) in the SPP column contributed to a lower longitudinal diffusion (hb). In contrast, the mobile phase mass transfer coefficient was similar in the two columns leading to comparable hc,m contributions. This study discloses some options for improvement of the efficiency of ZWIX-type chiral columns such as replacing narrow pore (120 A) by wide pore (200 A) particles, substituting FPP by SPP and reducing the selector density on the surface.

The present work reports on the preparation of polythiol-functionalized silica particles by thermally and photo-initiated radical addition reactions using poly(3-mercaptopropyl)methylsiloxane (PMPMS) as sulfhydryl group-rich surface modification reagent. Prior to surface modification with PMPMS, the silica was vinylized with vinyl trimethoxysilane. Finally, the usefulness of the thiolated silica particles was demonstrated by their further modification for various HPLC applications such as argentation chromatography and chiral separations. Aiming at a sulfhydryl group-rich, thin PMPMS layer on the surface of the silica several factors such as quantity of PMPMS, radical starter and reaction time were investigated by a design of experiment (DoE) approach. In thermally induced polymerization reactions 2,2'-azobis(isobutyronitrile) (AIBN) was used as radical starter, in photo-induced reactions 2,2-dimethoxy-2-phenylacetophenone (DMPA) was used instead. The incorporation of PMPMS was evaluated by elemental analysis and reactive and accessible sulfhydryl groups were determined by performing a thiol-disulfide exchange reaction with 2,2'-dipyridyl disulfide (DPDS). Consequently, thiol-functionalized silica particles (200 A, 5 µm) with 1.81 ± 0.07 µmol sulfhydryl groups per m2 were prepared and further functionalized for silver ion chromatography and chiral separation chromatography clearly proving its utility as platform for further silica functionalization. The fabricated stationary phase for silver ion chromatography showed promising separation abilities for fatty acid methyl esters (FAME) according to the amount of double bonds within the fatty acid residue and cis- and trans-stilbene as model molecule for cis-trans isomerism. After the successful incorporation of O-tert-butylcarbamoyl quinine (tBuCQN) as chiral selector via thiol-ene click chemistry onto the PMPMS layer, the obtained chiral stationary phases (CSP) showed good separation of derivatized amino acids in polar organic elution mode comparable with a column based on commercially available CHIRALPAK QN-AX silica particles (120 A, 5 µm).

A thin functional film of poly(3-mercaptopropyl)methylsiloxane was coated onto vinyl-modified silica particles (5μm, 100Å pore size) and chemically crosslinked to the surface. Excess of thiol functionalities allow bonding of alkene containing ligands by thiol-ene click reaction in a second step (QN-VII). Besides that a single step surface modification procedure was established in which alkene functional ligands were directly added to the polysiloxane coating solution and thus, after evaporation of the solvent, crosslinking to the vinylized surface and bonding of chromatographic ligand to the thiolated polysiloxane film occur simultaneously in one step (QN-VI). Successful bonding of the polysiloxane film was confirmed for both approaches by (29)Si cross-polarization/magic angle spinning NMR spectra. The new surface functionalization concept can be utilized as a new platform for the preparation of various low-bleed, mass spectrometry-compatible stationary phases with a variety of functional ligands. The concept was demonstrated by thiol-ene click reaction with quinine carbamate and its subsequent use for enantiomer separation by HPLC-UV and HPLC-ESI-QTOF-MS of acidic chiral analytes. Chromatographic enantioselectivities were similar to a comparable brush-type CSP (QN-V0). The greatly reduced background signal in LC-MS, however, comes at expense of somewhat lower chromatographic efficiencies (C-term by factor of 2 larger compared to brush-type CSP). For quantitative analysis in single reaction monitoring (MRM(HR)) in high sensitivity mode, limit of detection and limit of quantification results are comparable for both surface-polymer modified CSPs, with only slightly higher values for the conventional brush-type CSP (QN-V0).

This work deals with the preparation of single-labeled bioconjugates of the antibiotic Gentamicin (GT) with the sulforhodamine-derived fluorescence dye Texas Red(®)-X (TR), its purification by high-performance liquid chromatography (HPLC) and its characterization by high-resolution mass spectrometry. Aminoglycosides such as GT are efficient antibiotics, but also problematic due to severe side effects such as nephro- and ototoxicity. Fluorescent labeled GT is used to visualize cellular uptake and distribution of the antibiotic to finally understand the mechanisms of serious adverse drug reactions. Pharmaceutically administered GT is a mixture of mainly four different components, which exhibit three (GT(C1)) or four (GT(C1a), GT(C2), GT(C2a)) primary amino functional groups which can be coupled with the labeling reagent TR. Thus, multiple labeling could be envisaged which was assumed to be detrimental for uptake studies by fluorescence imaging. The proposed synthesis aimed at preparation of single labeled product and together with the employed purification strategy indeed yielded single labeled GT as product. Analytical control of the reaction product was carried out by means of mass spectrometry (UHPLC-ESI-QTOF-MS/MS) to rule out over-labeling of GT, which would alter the physicochemical characteristics of GT and its cellular uptake significantly. Moreover, LC-MS/MS analysis gave valuable insights into structural diversity of single labeled products. Further, high-resolution MS and MS/MS spectra of underivatized GT are provided as well. The analytical information on preparation strategy and structure diversity is valuable for studies with a clinical focus on research of aminoglycoside toxicity. Furthermore, it is deemed to be useful for the development of LC-MS/MS assays for the determination of aminoglycosides or the fast screening of synthetic biology samples from biotechnological drug discovery.

Oxylipins play an important role in cell signaling and they act as auto- and paracrine factors. There are numerous reports on the analysis of oxylipins in biofluids, especially in plasma. Only a limited number of studies addressed the analysis of oxylipins in platelets using modern, sensitive LC MS methods, even though these compounds have a huge impact on platelet functions and thrombo-inflammation. In this work, a new method based on superficially porous particle (2.7 μm) capillary column (0.5 mm ID) and micro-liquid chromatography coupled to tandem mass spectrometry (μUHPLC-ESI-QqQ-MS/MS) method has been developed, optimized and validated. It has finally been successfully applied for human plasma and platelet analysis. The method allows the precise and accurate simultaneous quantification of 42 oxylipins with 13 deuterated internal standards. Solid phase extraction with Bond Elut Certify II provides good extraction recoveries (on average around 75 %). The μUHPLC-MS/MS method is selective, sensitive (LOQs between 30 and 150 pg/mL) and shows good linearity. Limits of detections for most of the compounds are between 2 and 250 fmol on column. Twenty-three oxylipins have been detected in plasma and 19 in non-activated (resting) platelets (all samples were from healthy donors). The μUHPLC-MS/MS method uses very low volume of mobile phase (less than 250 μL of organic solvents in mobile phase per analysis), and therefore is considered environmentally friendly. It also turned out to be robust enough for routine analysis.

A new commercially available HPLC column, poly-N-(1H-tetrazole-5-yl)-methacrylamide-bonded stationary phase (Daicel DCpak PTZ), was systematically evaluated for its carbohydrate isomer separation capability by hydrophilic interaction liquid chromatography (HILIC) with charged aerosol detection (CAD) or (tandem) mass spectrometry. Reducing sugars tend to split into two anomer peaks which makes carbohydrate isomer separations in non-derivatized form even more complicated. For practical purposes anomer separations are therefore ideally suppressed which can be accomplished by using high temperature or high pH that are both associated with fast interconversion kinetics leading to peak coalescence, or on the other hand by conditions with low chromatographic anomer selectivity. Four major hexoses (glucose, mannose, galactose, fructose), five main pentoses (ribose, ribulose, xylose, xylulose, arabinose) and five most important disaccharides (maltose, cellobiose, lactose, sucrose, trehalose) were analyzed as single carbohydrate standards by isocratic HILIC with 0.1% (v/v) formic acid and 2 mM ammonium acetate at various temperatures to study anomer interconversion equilibria in a pH-dependent manner. Rate constants of forward (α→β) and backward (β→α) anomerization and corresponding energy barriers were calculated. The energy barriers of anomerisation were in the range of around 83–91 kJ mol−1 at 298 K and the difference between forward (α→β) and backward reaction (β→α) was typically between 1–3 kJ mol−1. The systematic studies finally allowed to pick conditions for the simultaneous analysis of all 14 carbohydrates by HILIC-ESI-MS(/MS) with PTZ in gradient elution mode. A combination of carbohydrate isomer-selective LC (with PTZ), tandem MS (with carbohydrate group-selective MS1 and some species-specific SRM transitions) and a simple deconvolution strategy allowed the determination of all carbohydrates of the complex test mixture except for the disaccharide pair lactose and maltose (which can be determined as sum). Consequently, the proposed method represents a successful step towards a global glycometabolomics profiling method of mono- and disaccharides by HILIC-ESI-MS/MS.

A cardinal requirement for effective 2D-HPLC separations is sufficient complementarity in the retention profiles of first and second dimension separations. It is shown that retention and enantioselectivity of chiral selectors derived from cinchona alkaloids can be conveniently modulated by structural variation of the carbamate residue of the quinine/quinidine carbamate ligand of such chiral stationary phases (CSP). A variety of aliphatic and aromatic residues have been tested in comparison to non-carbamoylated quinine CSP. Various measures of orthogonality have been utilized to derive the CSP that is most complementary to the tert-butylcarbamoylated quinine CSP (tBuCQN CSP), which is commercially available as Chiralpak QN-AX column. It turned out that O-9-(2,6-diisopropylphenylcarbamoyl)-modified quinine is most promising in this respect. Its implementation as a complementary CSP for the separation of amino acids derivatized with Sanger’s reagent (2,4-dinitrophenylated amino acids) in the first dimension combined with a tBuCQN CSP in the second dimension revealed successful enantiomer separations in a comprehensive chiral×chiral 2D-HPLC setup. However, the degree of complementarity could be greatly enhanced when simultaneously the absolute configurations were exchanged from quinine to quinidine in the chiral selector of the first dimension separation resulting in opposite elution orders of the enantiomers in the two dimensions. The advantage of such a chiral×chiral over achiral×chiral 2D-HPLC setup, amongst others, is the perfect compatibility of the mobile phase because in both dimensions the identical eluent can be used.

In spite of demonstrated lack of accuracy and consistency, quantification of steroid hormones is still most commonly executed via immunoassays. Mass spectrometric methods with triple quadrupole instruments are well established and, because of their proven robustness and sensitivity, best suited for targeted analysis. However, recent studies have shown that high-resolution mass spectrometers, like quadrupole time-of-flight instruments (QTOF), show comparable performance in terms of quantification and can generate additional sample information via untargeted profiling workflows. We demonstrate that adequate accuracy and selectivity for estradiol and testosterone can be achieved with a QTOF by data-independent acquisition with sequential window acquisition of all theoretical fragment-ion mass spectra (SWATH). Besides potential combination of targeted quantification and untargeted profiling, SWATH offers advantages with respect to sensitivity because the reduced total number of MS/MS experiments could be used to increase accumulation time without increasing cycle time. By applying a surrogate calibrant method leading to successful validation, a reliable method for absolute steroid quantification and high potential for steroid profiling has been developed. Linear calibration was achieved in the range from 10 to 1,000 pg mL−1 for 13C3-estradiol and from 20 to 15,000 pg mL−1 for 13C3-testosterone. Results for inter-day precision (13C3-estradiol: 4.5–10.2%; 13C3-testosterone: 5.1–7.8%) and inter-day accuracy (13C3-estradiol: 94.6–112.8%; 13C3-testosterone: 98.2–107.7%) were found to be well acceptable. Eventually, the method has been utilized to measure clinical samples of a study in which male volunteers obtained transdermal estradiol patches and sex hormone levels were quantified in plasma.

The ability to accurately quantify the protein coverage on nanoparticles is critical for assessing the quality of the surface chemistry and the success of the functionalization process of protein-nanoparticle conjugates. Surface coverage determination is therefore an integral part in the quality control of protein-modified nanoparticles in industrial nanotechnology. In this work, a novel and conventional method was established for direct quantification of the protein surface coverage on metallic nanoparticles. Different concentrations of pepsin were conjugated to gold nanoparticles (GNPs) by a straightforward adsorptive immobilization process as a model system, and a protein quantitation methodology based on the amino acid analysis of the hydrolysate of the protein-GNP conjugates was established. For this purpose, pepsin functionalized GNPs (pepsin-GNP bioconjugates) were processed via in situ hydrolysis with 6N HCl and subsequent derivatization with 6-aminoquinolyl- N -hydroxysuccinimidyl carbamate (AQC reagent). Direct quantitative amino acid analysis was performed based on measuring the intensity of AQC-glycine derivative by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). The method allows for detection of surface coverages as low as 0.1 μg mL −1 pepsin (corresponding to 2.89 × 10 −9 mol L −1 ) in the colloidal solution. Method imprecision for replicated surface coverage determinations was

This article provides a condensed introduction to principles of chiral separation, gives a historic overview of the genesis of the most important concepts regarding chiral stationary phase (CSPs), and summarizes the state of the art in a concise manner. Some recent developments in the field of polysaccharide CSPs are outlined. Finally, the article focusses on the new concept of zwitterionic chiral stationary phases and their application profile and peculiarities. Some other trends in column technology, including sub-2 μm and core-shell CSP particles and the emerging field of (UP)SFC, are briefly discussed.

In the present contribution we report a HPLC enantioseparation method for a library of amido-aminophosphonate structures generated by a novel Ugi-multicomponent reaction. The enantioseparation of these novel potentially bioactive molecules was achieved by means of HPLC on cinchona-carbamate based chiral anion-exchangers under polar organic elution conditions. The compounds were synthesized with the aim of offering a wide degree of heterogeneity in terms of structural elements to evaluate the influence of various structural motifs on enantioselectivity. The resultant compound library allows to derive structure-enantioselectivity relationships useful for understanding the chiral recognition process. As part of the study mobile phase characteristics such as concentration and type of counterion, mobile phase composition, apparent pH and temperature were investigated with regards to their effect on the separation performances. Employing a single mass spectrometry-compatible mobile phase, 23 compounds out of a pool of 42 racemic amido-aminophosphonate structures reached full baseline separation (Rs1.5) and 5 were almost baseline separated (1Rs1.5). This result clearly demonstrates the wide applicability of carbamoylated-cinchona alkaloids as chiral stationary phases for this class of analytes.

We report a chiral high-performance liquid chromatographic enantioseparation method for free α-aminophosphonic, β-aminophosphonic, and γ-aminophosphonic acids, aminohydroxyphosphonic acids, and aromatic aminophosphinic acids with different substitution patterns. Enantioseparation of these synthons was achieved by means of high-performance liquid chromatography on CHIRALPAK ZWIX(+) and ZWIX(-) (cinchona-based chiral zwitterionic ion exchangers) under polar organic chromatographic elution conditions. Mobile phase characteristics such as acid-to-base ratio, type of counterion, and solvent composition were systematically varied in order to investigate their effect on the separation performance and to achieve optimal separation conditions for the set of analytes. Under the optimized conditions, 32 of 37 racemic aminophosphonic acids studied reached baseline separation when we employed a single generic mass-spectrometry-compatible mobile phase, with reversal of the elution order when we used (+) and (-) versions of the chiral stationary phase.

Plasmid DNA may exist in three isoforms, the linear, open-circular (oc, "nicked"), and covalently closed circular (ccc, "supercoiled") form. We have recently reported on the chromatographic separation of supercoiled plasmid topoisomers on cinchona-alkaloid modified silica-based stationary phases. Herein, we present a selectivity switching mechanism to achieve separation of isoforms and/or supercoiled topoisomers using the very same chromatographic column and system. While salt gradient elution facilitates topoisomer separation, the supercoiled species are eluting as a single peak upon elution by a mixed pH and organic modifier gradient, still well separated from the other isoforms. We have found that a mobile phase pH value near the pI of the zwitterionic adsorbent surface leads to full recovery of all plasmid DNA isoforms, which is a major issue when using anion exchange-based resins. Furthermore, the observed elution pattern, oc < linear < ccc, is constant upon changes of mobile phase composition, gradient slope, and plasmid size. The remarkable isoform selectivity found on quinine-based selectors is explained by van't Hoff plots, revealing a different binding mechanism between the supercoiled plasmid on one hand and the oc and linear isoforms on the other hand.

This work reports on the proof-of-principle of preparation of novel one step in-situ functionalized monolithic polysiloxane-polymethacrylate composite materials in capillary columns for enantioselective nano-HPLC using a thiol-ene click reaction. Quinine carbamate as functional monomer and ethylene dimethacrylate as crosslinker were both used as ene components in a thermally initiated double click-type polymerization reaction with poly(3-mercaptopropyl)methylsiloxane as thiol component in presence of 1-propanol as porogenic solvent. Elemental analysis and on-capillary fluorescence measurement proved the successful incorporation of the functional chiral monomer into the polymer. Scanning electron microscopy images revealed a macroporous polymer morphology which is typical for a nucleation and growth mechanism of pore formation. The individual microglobules appear relatively spherical and smooth indicating a non-porous nature. Nano-HPLC experiments of the chiral monolithic capillary column provided successful enantiomer separation of N-3,5-dinitrobenzoylleucine as test compound in polar organic elution mode clearly documenting the successful implementation of the proposed concept towards new functionalized monolithic composite materials.

Immobilization of enzymes on mesoporous microparticulate carriers has traditionally been accompanied by reduction in enzyme activity. Herein, we document that immobilization of pepsin via amide coupling on gold nanoparticles (GNPs) with a carboxy-terminated hydrophilic PEG7 shell resulted in a heterogeneous nanobiocatalyst with essentially equivalent turnover rates, k cat (90 %), and enhanced catalytic efficiencies, k cat/K M (107 %), compared to homogeneous catalysis with pepsin in free solution for cytochrome C as model substrate. This heterogeneous catalyst showed further at least equivalent bioactivity in a digestion reaction of a protein mixture consisting of cytochrome C, bovine serum albumin, and myoglobin. UHPLC-ESI-QTOF-MS/MS analysis of the digests with subsequent Mascot database search allowed unequivocal identification of all proteins with high score and good sequence coverage. The functionalized nanoparticles were further characterized by Vis spectroscopy in terms of the surface plasmon resonance (SPR) band, by dynamic light scattering (DLS) with regard to hydrodynamic diameters, and in view of their ζ potentials at each step of synthesis and surface modification. These measurements also revealed that the pepsin-functionalized GNPs were sufficiently stable over at least 1 month; thus providing a satisfactory shelf life to the heterogeneous catalyst. Advantageously, the pepsin-GNP bioconjugate can be conveniently removed after reaction by simple centrifugation steps which makes them a useful tool for analysis of therapeutic peptides and proteins, including monoclonal antibodies. The practical utility of the nanobiocatalyst was documented by digestion of a monoclonal antibody which yielded the F(ab')2 fragment with a mass of 97,619.4 Da. Graphical Abstract Pepsin conjugated to pegylated gold nanoparticles exhibit properties of homogeneous catalysis such as enzyme activity like pepsin in solution and ability to pipette the stable colloidal suspension whereas one can take benefit from easy removal by centrifugation and re-use which are characteristics of heterogeneous catalysis.

Plasmid DNA (pDNA) holds great potential for use in genetherapy and DNA vaccines. Therefore much attention hasbeen paid to the biotechnological production of novel drugsof this type as they enter clinical evaluation. Herein wepresent a unique liquid-chromatography-based method forthe efficient separation of topoisomers of the covalentlyclosedcircular(ccc)plasmidDNA,whichisconsideredbytheU.S. Food and Drug Administration to be therapeutically themost active isoform.

Chiral alpha-arylthiocarboxylic acids with different substitution patterns, representing new pirinixic acid derivatives with dual PPARalpha/gamma agonistic activities, have been separated into enantiomers on tert-butylcarbamoylquinine and quinidine based chiral anion-exchangers and amylose tris(3,5-dimethylphenylcarbamate) coated silica on analytical and preparative scale. Absolute configurations of individual enantiomers were assigned chromatographically via elution orders on the chiral anion-exchangers and were confirmed by stereoselective syntheses via Ewans auxiliaries that have lead to enantiomeric products with known absolute configurations. The results of both methods were in full agreement. Moreover, the receptor stereoselectivity in PPARalpha transactivation activities was consistent within the test set of structurally related compounds. Limited correlation (between elution order and substitution) was observed within the set of alpha-arylthiocarboxylic acids on the amylose tris(3,5-dimethylphenylcarbamate) based chiral stationary phase (CSP), in particular the elution order changed with remote substitution. This clearly demonstrates the risks of chromatographic absolute configuration assignments by prediction from one structural analog to another one, especially with CSPs such as polysaccharide CSPs that are recognized for their broad applicability due to multiple binding and chiral recognition modes. It is therefore of utmost importance that such chromatographic absolute configuration predictions by extrapolation to structural analogs are combined with orthogonal methods for verification of the results.

The presented work deals with the development and comprehensive validation of a quantitative LC-electrospray ionization (ESI)-tandem mass spectrometry (MS/MS) method using a triple quadrupole instrument in the MRM mode for the metabolic profiling of amino acids, organic acids, vitamins, some biogenic amines, secondary metabolites of β-lactam antibiotics biosynthesis as well as their intermediates, and degradation products in fermentation broths of β-lactam antibiotics production (in total 57 hydrophilic compounds). A great number of chromatographic systems (22 different stationary phase/mobile phase conditions) were screened for their adequate chromatographic selectivity to cope with isobaric compounds and other critical analyte pairs. Finally, a hydrophilic interaction liquid chromatography (HILIC) method employing a zwitterionic ZIC-HILIC column was selected as best compromise. Particular focus was given on the elucidation of absolute and relative matrix effects via comparison of slopes of calibration functions of spiked matrix and standard solutions. These data as well as precision and accuracy data confirm suitability of the HILIC-ESI-MS/MS assay for metabolic profiling studies in fermentation samples. Detailed comprehensive data sets are presented which should illustrate critical issues, problems, and challenges of multitarget quantitative metabolic profiling and should outline possible strategies to circumvent pitfalls and overcome common problems.