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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

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.

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.

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.

Amino acid analysis (AAA) is of central importance for the characterization of the amino acid composition of proteins, peptides, pharmaceutical formulations, dietary supplements, in bioanalysis and metabolomics. Common methodologies are based on achiral assays and hence do not consider distinction of D and L-amino acid enantiomers. This may be misleading in many instances, because therapeutic peptides and natural peptides synthesized by non-ribosomal peptide synthetases (NRPSs) frequently contain D-amino acids to achieve proteolytic stability. Furthermore, stereochemical integrity control of peptides made from L-amino acids also need stereoselective assays, and racemization in natural products (proteins, peptides, amino acids) have shown to be useful biomarkers of disease and require assays, which can provide information about the individual stereoisomers. Hence, rapid enantioselective amino acid analysis (ReAAA) is of utmost importance. However, currently employed enantioselective assays are often only focusing on a limited number of amino acids or require long analysis times making them incompatible for high-throughput sample analysis. Here, we present an ReAAA assay, which is based on a fast UHPLC enantiomer separation using a short QN-AX core-shell particle column (2.7 µm) coupled to electrospray ionization quadrupole time of flight mass spectrometric (ESI-QTOF-MS) detection. Amino acid samples are mixed with a [u-13C15N]-labelled L- or DL-amino acid standard mixture, precolumn derivatized with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent and subjected to UHPLC-ESI-MS analysis. A number of chromatographic variables have been optimized (buffer additives and concentration, water content, flow rate and temperature) to achieve the goal of simultaneous amino acid enantiomer separation of all proteinogenic amino acids within the shortest possible analysis time. A mobile phase consisting of 50 mM NH4FA, 50 mM FA and 0.5% H2O in MeOH combined with a flow rate of 1 mL/min and a column temperature of 30 °C has been identified as optimal. With these conditions, all of the proteinogenic DL-enantiomers (except for Arg) can be separated in less than 2.5 min. If D-Arg and distinction between D-Leu/D-Ile is of concern, a second-tier method can accomplish this goal. The method can be very useful for ReAAA when high sample throughput is a major demand. Its utility was demonstrated by ReAAA of a lipopeptide hydrolysate which contained several D-amino acids and of an amino acid supplement in which minor D-amino acid impurities could be detected.