RP Chromatography Research Articles

Identification and Characterization of the Stability of Hydrophobic Cyclolinopeptides From Flaxseed Oil.

Flaxseed (linseed) is a cultivar of the spring flowering annual plant flax (Linum usitatissimum) from the Linaceae family. Derivatives of this plant are widely used as food and as health products. In recent years, cyclic peptides isolated from flaxseed and flaxseed oil, better known as cyclolinopeptides (CLPs), have attracted the attention of the scientific community due to their roles in the inhibition of osteoclast differentiation or their antimalarial, immunosuppressive, and antitumor activities, as well as their prospects in nanotechnology and in the biomedical sector. This study describes the detection, identification, and measurement of CLPs in samples obtained from nine different flaxseed oil manufacturers. For the first time, Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer was used for CLP identification together with RP-HPLC. The routine analyses were performed using RP chromatography, measuring the absorption spectra and fluorescence detection for identifying tryptophan-containing peptides using the native fluorescence of tryptophan. In addition, existing protocols used for CLP extraction were optimized and improved in a fast and cost-efficient way. For the first time, 12 CLPs were separated using methanol/water as the eluent with RP-HPLC. Finally, the stability and degradation of individual CLPs in the respective flaxseed oil were examined over a period of 60 days at different temperatures. The higher temperature was chosen since this might reflect the cooking practices, as flaxseed oil is not used for high-temperature cooking. Using HPLC–MS, 15 CLPs were identified in total in the different flaxseed oils. The characterization of the peptides via HPLC–MS highlighted two types of CLP profiles with a substantial variation in the concentration and composition of CLPs per manufacturer, probably related to the plant cultivar. Among the observed CLPs, CLP-O, CLP-N, and CLP-B were the least stable, while CLP-C and CLP-A were the most stable peptides. However, it is important to highlight the gradual degradation of most of the examined CLPs over time, even at room temperature.

Metabolomic Profiling of Human Urine Samples Using LC-TIMS-QTOF Mass Spectrometry.

The identification of metabolites in biological samples is challenging due to their chemical and structural diversity. Ion mobility spectrometry (IMS) separates ionized molecules based on their mobility in a carrier buffer gas giving information about the ionic shape by measuring the rotationally averaged collision cross-section (CCS) value. This orthogonal descriptor, in combination with the m/z, isotopic pattern distribution, and MS/MS spectrum, has the potential to improve the identification of molecular molecules in complex mixtures. Urine metabolomics can reveal metabolic differences, which arise as a result of a specific disease or in response to therapeutic intervention. It is, however, complicated by the presence of metabolic breakdown products derived from a wide range of lifestyle and diet-related byproducts, many of which are poorly characterized. In this study, we explore the use of trapped ion mobility spectrometry (TIMS) via LC parallel accumulation with serial fragmentation (PASEF) for urine metabolomics. A total of 362 urine metabolites were characterized from 80 urine samples collected from healthy volunteers using untargeted metabolomics employing HILIC and RP chromatography. Additionally, three analytes (Trp, Phe, and Tyr) were selected for targeted quantification. Both the untargeted and targeted data was highly reproducible and reported CCS measurements for identified metabolites were robust in the presence of the urine matrix. A comparison of CCS values among different laboratories was also conducted, showing less than 1.3% ΔCCS values across different platforms. This is the first report of a human urine metabolite database compiled with CCS values experimentally acquired using an LC-PASEF TIMS-qTOF platform.

Urinary metabolomic signature of muscle-invasive bladder cancer: A multiplatform approach

Bladder cancer (BCa) is ninth amongst the most common types of cancer in the human population worldwide. The statistics of incidence and mortality of BCa are alarming and the currently applied diagnostic methods are still not sensitive enough. This leads to a large number of undiagnosed BCa cases, usually among patients in the early stages of the disease. Despite the fact that many risk factors of BCa have been recognized, the pathomechanism of development of bladder cancer has not been fully explained yet. Therefore, in the present study, multiplatform urinary metabolomics has been implemented in order to scrutinize potential diagnostic indicators of BCa that might help to explain its pathomechanism and be potentially useful in diagnosis and determination of stage of the disease. Urine samples collected from muscle-invasive high grade BCa patients (n = 24) and healthy volunteers (n = 24) were matched in terms of most common BCa risk factors i.e. gender, age, BMI and smoking status. They were analyzed by high performance liquid chromatography coupled with time of flight mass spectrometry detection (HPLC-TOF/MS) using RP and HILIC chromatography, gas chromatography hyphenated with triple quadruple mass spectrometry detection (GC-QqQ/MS) in scan mode, and proton nuclear magnetic resonance (1H NMR). The six datasets obtained were submitted to univariate and multivariate statistical analyses. 17 metabolites significantly discriminated urinary profiles of BCa patients from urinary profiles of healthy volunteers. These metabolites are mainly involved in amino acid metabolism, pyrimidine and purine metabolism, as well as energy metabolism and might play a crucial role in the pathogenesis of BCa.

Merging metabolomics and lipidomics into one analytical run

A novel integrated metabolomics/lipidomics workflow is introduced enabling high coverage of polar metabolites and non-polar lipids within one analytical run. Dual HILIC and RP chromatography were combined to high-resolution mass spectrometry. As a major advantage, only one data file per sample was obtained by fully automated simultaneous analysis of two extracts per sample. Hence, the unprecedented high coverage without compromise on analytical throughput was not only obtained by the orthogonality of the chromatographic separations, but also by the implementation of dedicated sample preparation procedures resulting in optimum extraction efficiency for both sub-omes. Thus, the method addressed completely hydrophilic sugars and organic acids next to water-insoluble triglycerides. As for the timing of the dual chromatography setup, HILIC and RP separation were performed consecutively. However, re-equilibration of the HILIC column during elution of RP compounds and vice versa reduced the overall analysis time by one third to 32 min. Application to the Standard Reference Material SRM 1950 - Metabolites in Frozen Human Plasma resulted in >100 metabolite and >380 lipid identifications based on accurate mass implementing fast polarity switching and acquiring data dependent MS2 spectra with the use of automated exclusion lists. Targeted quantification based on external calibrations and 13C labeled yeast internal standards was successfully accomplished for 59 metabolites. Moreover, the potential for lipid quantification was shown integrating non-endogenous lipids as internal standards. In human plasma, concentrations ranging over 4 orders of magnitude (low nM to high μM) were assessed.

Tandem HILIC-RP liquid chromatography for increased polarity coverage in food analysis.

Comprehensive non-targeted analysis of food products normally requires two complementary chromatographic runs to achieve maximum compound coverage. In this study, we present a sensitive tandem-LC method, which combines RP and HILIC separation in a single run. The setup consists of a C18 trap column and two subsequently coupled analytical columns (HILIC and C18) which are operated in parallel. First, hydrophobic compounds are retained on the RP trap column while rather hydrophilic compounds are directly transferred onto a HILIC phase. Next, the pre-fractionated sample composition is analyzed by HILIC or RP chromatography, respectively. The presented setup allows individual and independent gradient elution as well as interfacing with mass spectrometry. The performance of the method has been proven by means of food relevant standards and analysis of complex food samples (e.g. red wine, meat extract). The simple and robust setup provides high flexibility in the selection of column combinations and does not require sophisticated instrumental setups or software. The method significantly increases the covered polarity range compared to classical one-dimensional chromatography. Our results indicate that tandem LC is a valuable and universal tool in the non-targeted screening of various types of complex food samples.

Direct injection of HILIC fractions on the reversed-phase trap column improves protein identification rates for salivary proteins.

Online combination of hydrophilic interaction chromatography (HILIC) and RP chromatography for separation of tryptic peptides is a challenging approach due to the incompatibility of direct loading HILIC fractions on the RP trapping column. High amounts of organic modifiers in loading solvents decrease the binding efficiency of tryptic peptides on C18 phases and lower the number of identifications. A 500 μL loop upfront of the trapping column filled with aqueous mobile phase was employed as a mixing chamber and enabled direct injections and improved saliva protein identification rates of HILIC fractions.

Preparation of phosphorylcholine-based hydrophilic monolithic column and application for analysis of drug-related impurities with capillary electrochromatography.

A hydrophilic monolithic CEC column was prepared by thermal copolymerization of zwitterionic monomer 2-methacryloyloxyethyl phosphorylcholine (MPC), pentaerythritol triacrylate (PETA), either methacrylatoethyl trimethyl ammonium chloride (META) or sodium 2-methylpropene-1-sulfonate (MPS) in a polar binary porogen consisting of methanol and THF. A typical hydrophilic interaction LC retention mechanism was observed for low-molecular weight polar compounds including amides, nucleotides, and nucleosides in the separation mode of hydrophilic interaction CEC, when high content of ACN (>60%) was used as the mobile phase. The effect of the electrostatic interaction between the analytes and the stationary phase was found to be negligible. The poly(MPC-co-PETA-co-META or MPS) monolithic columns have an average column efficiency of 40000 plates/m and displayed with a satisfactory repeatability in terms of migration time and peak areas. Finally, the column was successfully applied to determine the impurities of a positively charged drug pramipexole which are often separated by ion pair RP chromatography due to their high hydrophilicity. All four components can be baseline separated within 5 min with BGE consisting of ACN/20mM ammonium formate buffer (pH 3.0; 80/20).

Glutathione analogues as substrates or inhibitors that discriminate between allozymes of the MDR-involved human glutathione transferase P1-1.

Glutathione (GSH) structure-guided tripeptide analogues were designed and synthesized by solid phase technology, purified (≥95%) by RP and/or GF column chromatography, to identify those that, compared with GSH, exhibited similar or higher binding and catalytic efficiency toward the MDR-involved human GSTP1-1 isoenzyme, and could discriminate between the allozymic expression products of the polymorphic human GSTP1 gene locus, designated as hGSTP1*A (Ile(104) /Ala(113) ), hGSTP1*B (Val(104) /Ala(113) ), and hGSTP1*C (Val(104) /Val(113) ). The analogues bear single amino acid alterations as well as alterations in more than one position. Some analogues showed remarkable allozyme selectivity, binding catalytically to A (I, II, IV, XII), to C (V and XVI), to A and C (III, VII, XIV) or to all three allozymes (XV). A heterocyclic substituent at positions 1 or 2 of GSH favors inhibition of A, whereas a small hydrophobic/hydrophilic amide substituent at position 2 (Cys) favors inhibition of B and C. Heterocyclic substituents at position 1, only, produce catalytic analogues for A, whereas less bulky and more flexible hydrophobic/hydrophilic substituents, at positions 1 or 3, lead to effective substrates with C. When such substituents were introduced simultaneously at positions 1 and 3, the analogues produced have no catalytic potential but showed appreciable inhibitory effects, instead, with all allozymes. It is anticipated that when GSH analogues with selective inhibitory or catalytic binding, were conjugated to allozyme-selective inhibitors of hGSTP1-1, the derived leads would be useful for the designing of novel chimeric inhibitors against the MDR-involved hGSTP1-1 allozymes. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 330-344, 2016.

Isotype-specific glycosylation analysis of mouse IgG by LC-MS.

With mice being the top model organism in immunology and with Fc glycosylation being increasingly recognized as important modulator of antibody function, the time has come to take a look at the glycosylation of mouse IgG isotypes. Tryptic glycopeptides of mouse IgG1, IgG2, and IgG3 differ in mass and so these three isoforms can be easily discriminated by MS. Commercial IgG contained a rare IgG1 variant but no IgG3, which, however, was found in sera of C57BL/6 and BALB/c mice. These strains deviated with regard to IgG2a and IgG2b alleles. The Ig2a B allele was not observed in any of the four samples investigated. All a/c isotypes contain the same glycopeptide sequence, which deviates from that of IgG2b by containing Leu instead of Ile. The Leu/Ile glycopeptide variants were separated by RP chromatography and the order of elution was determined. The major glycoforms on all isotypes were fucosylated with no and one galactose (GnGnF and GnAF) followed by fully galactosylated AAF and smaller amounts of mono- and disialylated N-glycans. In the commercial serum pool, the relative ratios of glycans differed between isotypes. Sialic acid exclusively occurred as N-glycolylneuraminic acid. Fucosylation was essentially complete. No bisected and no α1,3-galactosylated glycans were found.

Inversion of type of separation system in planar chromatography of peptides, using C18 silica-based adsorbents

Our previous results show, that C18 silica-based adsorbents used in high-performance thin-layer chromatography (HPTLC), provide complex retention mechanism basing on various polar and nonpolar interactions. Here we present, that in chromatography of peptides, due to mixed-mode properties of these adsorbents, there is a simple way to obtain inversion of separation system type (from reversed-phase, RP, to normal-phase, NP, and vice versa). The results presented provide detailed information how to obtain inversion mentioned and reflect the extent (the type and concentration of organic solvent, the type and concentration of ion-pairing reagent in the mobile phase) of this phenomenon. We show, that the system type inversion results in significant change of selectivity of separation, which may be especially useful in 2D separation of complex samples of basic/amphoteric compounds such as peptides. This results from the fact, that C18 silica-based HPTLC adsorbents, may be used in hydrophilic interaction chromatography (HILIC) or RP chromatography, in dependence on mobile phase composition.

Metabolite targeting: development of a comprehensive targeted metabolomics platform for the assessment of diabetes and its complications

Biomarker studies for metabolic disorders like diabetes mellitus (DM) are an important approach towards a better understanding of the underlying pathophysiological mechanisms of diseases (Roberts and Gerszten in Cell Metab 18:43–50, 2013; Wilson et al. in Proteome Res 4:591–598, 2005). Furthermore, screening of potential metabolic biomarkers opens the opportunity of early diagnosis as well as therapy and drug monitoring of metabolic disorders (Rhee et al. in J Clin Invest 10:1–10, 2011; Wang et al. in Nat Med 17:448–458, 2011; Wenk in Nat Rev Drug Discov 4:594–610, 2005). The aim of the present study was to develop methods for the quantitative determination of 74 potential metabolite biomarkers for DM and diabetic nephropathy (DN) in serum. Several studies have shown that the concentrations of many polar metabolites like amino or organic acids are changed in subjects suffering from diabetes (Wang et al. in Nat Med 17:448–458, 2011; Yuan et al. in J Chromatogr B 813:53–58, 2007). Analyzing polar analytes presents a challenge in liquid chromatography (LC) coupled with ESI–MS/MS (Gika et al. in J Sep Sci 31:1598–1608, 2008; Spagou et al. in J Sep Sci 33:716–727, 2010). Considering those reasons we decided to develop a specific HILIC–ESI–QqQ–MS/MS-method for quantitative determination of these polar metabolites. A subsequent method validation was carried out for both HILIC and RP chromatography with respect to the guidelines of the Food and Drug Administration (FDA in Food and Drug Administration: Guidance for industry, bioanalytical method validation, 2001). The HILIC and RP LC–MS methods were successfully validated. Furthermore, the HILIC method presented here was applied to serum samples of GIPRdn transgenic mice, a diabetic strain developing DN, and non transgenic littermate controls. Significant, diabetes-associated changes were observed for the concentrations of 21 out of 62 metabolites. The new methods described here accurately quantify 74 metabolites known to be regulated in diabetes, allowing for direct comparison between studies and laboratories. Thus, these methods may be highly adoptable in clinical research, providing a starting point for early diagnosis and metabolic screening.

A New N-Acyl Taurine from the South China Sea Marine Sponge Callyspongia sp.

The genus Callyspongia (order Haplosclerida, family Callyspongiidae) is widely distributed and contains various bioactive constituents [1]. In the course of our investigation on the marine sponge Callyspongia species, we have reported some metabolites [2, 3]. Further investigation on the marine sponge Callyspongia species has led to the isolation and characterization of a new N-acyl taurine (1). The sponge Callyspongia sp. was collected off the coast of Hainan Island, South China Sea, in January, 2007. The specimen was identified by Dr. Kyung Jin Lee. A voucher specimen (No. 20070101) was deposited in Natural History Museum, Hannam University, Daejon, Korea and Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology, Chinese Academy of Sciences, China. The wet marine sponge Callyspongia sp. (10 kg) was extracted with ethanol (90%). The ethanol was evaporated in vacuo to afford a syrupy residue that was suspended in distilled water and fractionated successively with petroleum ether, ethyl acetate, and n-butanol. The n-butanol soluble portion (80.1 g) was subjected to RP chromatography eluted with H2O–MeOH (9:1, fr. A), H2O–MeOH (8:2, fr. B), H2O–MeOH (7:3, fr. C), H2O–MeOH (6:4, fr. D), H2O–MeOH (4:6, fr. E), and H2O–MeOH (0:10, fr. F), to give six fractions. Fraction C (4.12 g) was subjected to silica gel chromatography with CHCl3–MeOH (8:2 to 6:4, gradient) to give three fractions (C1–C3). Fraction C2 (435 mg) was subjected to silica gel chromatography with CHCl3–MeOH (8:2), then purified by Sephadex LH-20 (CHCl3–MeOH, 1:1) to afford 1 (5.0 mg). Compound 1 was obtained as a yellow oil, and its molecular formula was deduced to be C13H25NO4S on the basis of the HR-ESI-MS spectrum at m/z 290.1422 [M – H]– (calcd 290.1426 for C13H24NO4S) and NMR data. The gross structure was determined by the aid of COSY, HMQC, and HMBC experiments. The 1H NMR, 13C NMR, and DEPT data for 1 indicated the presence of two methyls, eight sp3 methylenes, one sp2 methine, and two sp2 quaternary carbons. The sulfur and remaining three oxygens in the molecular formula suggested a sulfonic acid group (SO3H). The 1H NMR signals at 3.63 (2H, t, J = 7.0 Hz, H-1 ) and 3.00 (2H, t, J = 7.0 Hz, H-2 ), and 13C NMR signals at C 174.6 (C-1), 36.6 (C-1 ) and 51.5 (C-2 ), together with the HMBC correlations (Fig. 1) of H-2 with C-1 and H-2 with C-1 and C-1 , indicated the two methylenes C-1 and C-2 to be located between the amide and the sulfonic acid, which revealed a taurine moiety in the structure of 1 [4, 5]. The position of the double bond was ascertained at C-3 and C-4 on the basis of the HMBC correlations of H-4 with C-2 and H-5 with C-3. The methyl group (C-11) was determined to be located at C-3 by the HMBC correlations of H-11 with C-2 and C-4. The geometry of the double bond was deduced to be cis based on the chemical shifts of the allylic carbon C-5 at 29.2, which agreed well with the chemical shift for the allylic carbons of cis-olefins (approximately 29) [6]. Thus, the structure of compound 1 was elucidated as 2-(3-methyl-dec-3-enamido)ethanesulfonic acid. 2-(3-Methyl-dec-3-enamido)ethanesulfonic Acid (1). Yellow oil. 1H NMR (500 MHz, CDCl3, , ppm, J/Hz): 5.36 (1H, t, J = 6.5, H-4), 3.63 (2H, t, J = 7.0, H-1 ), 3.00 (2H, t, J = 7.0, H-2 ), 2.91 (2H, s, H-2), 2.07 (2H, m, H-5), 1.77 (3H, s, H-11), 1.18–1.27 (8H, overlap, H-6–9), 0.93 (3H, t, J = 6.5, H-10). 13C NMR (125 MHz, CDCl3, , ppm): 174.6 (C-1), 131.2 (C-4), 130.2 (C-3), 51.5 (C-2 ), 48.8 (C-2), 36.6 (C-1 ), 29.2 (C-5), 23.7–30.8 (C-6–9), 16.3 (C-11), 14.6 (C-10). HR-ESI-MS m/z 290.1422 [M – H]– (calcd 290.1426 for C13H24NO4S).

Integrating analytical resolutions in non-targeted wine metabolomics

Direct injection Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), and ultra-high performance liquid chromatography coupled to mass spectrometry (UPLC/MS) were combined for the non-targeted analysis of wine metabolites. The unrivalled resolution on mass measurement allowed by the former and the separation ability of isomeric and isobaric substances by the latter, clearly increases the scope of detectable unknown metabolites in wines. Such methodology is illustrated through the comparison of chemical spaces of a young and an older Pinot noir wine. RP and HILIC chromatography could reveal up to five isomers for a given mass, throughout the explored mass range. CHO, CHOS and CHONS chemical spaces exhibited higher diversities in the older wine, illustrating a molecular oeno-diagenesis process during wine ageing, whereas nitrogen-containing compounds (CHON chemical space) appeared to be fewer in the older wine, likely due to precipitation. This methodological combination is a promising contribution to the wine metabolomics toolkit.

The stereoselective separation of serine containing peptides by zwitterionic ion exchanger type chiral stationary phases and the study of serine racemization mechanisms by isotope exchange and tandem mass spectrometry

The occurrence of d-amino acids (d-AAs) in higher-developed organisms in their free form, and within peptides and proteins, has been investigated with an increasing number of studies. Often the inversion of the stereochemical configuration of an individual amino acid drastically changes its biological activity. Alongside Asn and Asp, Ser is most prone to racemization within peptides. Specific enzymes catalyzing d-Ser generation and breakdown have been described. Hence, the applicability of enantioselective ZWIX(+)® and ZWIX(−)® chiral stationary phases (CSPs) to peptide separations was assessed and a set of 14 pairs of diastereomeric and enantiomeric Ser and Thr containing di-, tri- and tetra-peptides was chromatographically separated without prior hydrolysis to the individual amino acids. To a certain extent, RP chromatography also enabled the separation of peptide diastereomers. The ZWIX CSPs delivered chromatographic selectivities between 1.04 and 7.23, allowing a change of elution order by switching between the ZWIX(+) and the ZWIX(−) CSP. Coupling these highly selective chromatographic columns with an LTQ-Orbitrap XL™ mass spectrometer and performing high resolution MS2 measurements enabled us to investigate mechanistic aspects of chemically induced racemization of Ser embedded in short peptides. As reaction medium an alkaline aqueous solution (pH 12.3) was selected. Proton/deuterium exchange experiments provided evidence of a fast Cα proton exchange with simultaneous racemization. Additionally, 18O/16O exchange allowed the identification of an alternative, and somewhat retarded racemization via a reversible β-elimination and reintroduction of water at the hydroxymethyl side chain of Ser. This involved the intermediate generation of the prochiral didehydro alanine unit.

A single-run liquid chromatography mass spectrometry method to quantify neuroactive kynurenine pathway metabolites in rat plasma

Neuroactive metabolites in the kynurenine pathway of tryptophan catabolism are associated with neurodegenerative disorders. Tryptophan is transported across the blood–brain barrier and converted via the kynurenine pathway to N-formyl-l-kynurenine, which is further degraded to l-kynurenine. This metabolite can then generate a group of metabolites called kynurenines, most of which have neuroactive properties.The association of tryptophan catabolic pathway alterations with various central nervous system (CNS) pathologies has raised interest in analytical methods to accurately quantify kynurenines in body fluids. We here describe a rapid and sensitive reverse-phase HPLC–MS/MS method to quantify l-kynurenine (KYN), kynurenic acid (KYNA), 3-hydroxy-l-kynurenine (3HK) and anthranilic acid (AA) in rat plasma. Our goal was to quantify these metabolites in a single run; given their different physico-chemical properties, major efforts were devoted to develop a chromatography suitable for all metabolites that involves plasma protein precipitation with acetonitrile followed by chromatographic separation by C18 RP chromatography, detected by electrospray mass spectrometry. Quantitation range was 0.098–100ng/ml for 3HK, 9.8–20,000ng/ml for KYN, 0.49–1000ng/ml for KYNA and AA. The method was linear (r>0.9963) and validation parameters were within acceptance range (calibration standards and QC accuracy within ±30%).

Preparative separation of crocins and geniposide simultaneously from gardenia fruits using macroporous resin and reversed-phase chromatography

Gardenia fruits contain valuable natural food colorants including crocins (gardenia yellow) and geniposide. In this study, a process for the enrichment of crocins and geniposide simultaneously from gardenia fruits was developed using macroporous resin and RP chromatography. The performance of eight different types of macroporous resins was evaluated. Static absorption/desorption experiments revealed that LX60 possessed optimal separating capacity. Further dynamic absorption/desorption experiments on LX60 columns were conducted to obtain the optimal parameters. After one run treatment with LX60, the content of crocin-1 in gardenia yellow reached 29.6%, while geniposide in another fraction reached 83.4%. An extract of crocins was obtained from gardenia yellow in a second-stage separation using RP medium-pressure LC, with its color value to be 756 and the content of crocin-1 reaching 60.8%. The separation process was highly efficient, low cost, and compact, which may be informative for purifications of other natural products from complex plant extracts.

Comparison of mobile‐phase systems commonly applied in liquid chromatography‐mass spectrometry of nucleic acids

LC-MS represents an important technology for the qualitative and quantitative analysis of nucleic acids. For MS, ESI in negative ion mode is used. The chromatographic method of choice is ion-pair (IP) RP chromatography. Chromatographic separations are usually accomplished by gradients of an organic modifier in aqueous solutions of IP reagents. Commonly applied IP reagents are 2.3 mM triethylamine/400 mM 1,1,1,3,3,3-hexafluoro-2-propanol (TEA/HFIP, pH 7.0) and 10-25 mM cyclohexyldimethylammonium acetate (CycHDMAA, pH 8.4). Direct comparison of mass spectrometric performance of the two solvent systems revealed that the TEA/HFIP system offers better detection sensitivity than the CycHDMAA system. This is mainly attributable to the depletion of HFIP during droplet formation and solvent evaporation. Removal of the anionic counterion facilitates oligonucleotide ionization, and the oligonucleotides are desorbed as highly charged ions into the gas phase. TEA/HFIP-based mobile phases are recommended for developing quantitative assays targeting defined oligonucleotides. The CycHDMAA system allows the formation of cyclohexyldimethylammonium adducts. These adducts are cleaved in the gas phase, and this decomposition gives rise to charge state reduction. Ammonium adduct formation is of particular importance in preventing adducting with metal ions. Thus, adducts with metal ions are efficiently suppressed with CycHDMAA. For the TEA/HFIP system, however, such adducting represents a severe problem particularly if large oligonucleotides are analyzed. Thus, CycHDMAA-based mobile phases are recommended for qualitative assays such as LC-MS-based genotyping.

Multidimensional separation of tryptic peptides from human serum proteins using reversed-phase, strong cation exchange, weak anion exchange, and fused-core fluorinated stationary phases

Proteome profiling of crude serum is a challenging task due to the wide dynamic range of protein concentrations and the presence of high-abundance proteins, which cover >90% of the total protein mass in serum. Peptide fractionation on strong cation exchange, weak anion exchange in the electrostatic repulsion hydrophilic interaction chromatography (ERLIC) mode, RP C18 at pH 2.5 (low pH), fused-core fluorinated at pH 2.5, and RP C18 at pH 9.7 (high pH) stationary phases resulted in two to three times more identified proteins and three to four times more identified peptides in comparison with 1D nanoChip-LC-MS/MS quadrupole TOF analysis (45 proteins, 185 peptides). The largest number of peptides and proteins was identified after prefractionation in the ERLIC mode due to the more uniform distribution of peptides among the collected fractions and on the RP column at high pH due to the high efficiency of RP separations and the complementary selectivity of both techniques to low-pH RP chromatography. A 3D separation scheme combining ERLIC, high-pH RP, and low-pH nanoChip-LC-MS/MS for crude serum proteome profiling resulted in the identification of 208 proteins and 1088 peptides with the lowest reported concentration of 11 ng/mL for heat shock protein 74.

Highly stable surface modification of hypercrosslinked monolithic capillary columns and their application in hydrophilic interaction chromatography

A two-step surface modification of poly(styrene-co-vinylbenzyl chloride-co-divinylbenzene) monolithic stationary phases, including hypercrosslinking and thermally initiated surface grafting of [2-(methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)ammonium hydroxide, has been used to prepare capillary columns for the isocratic separation of small polar compounds in hydrophilic interaction chromatography (HILIC). The prepared monolithic columns provided a dual retention mechanism, combining RP and hydrophilic interaction chromatography, controlled by the composition of the mobile phase. By the optimization of the surface grafting reaction using response surface methodology, we have found that the grafting time is the main synergistic effect controlling the retention of polar compounds in HILIC. The prepared monolithic columns achieved long column lifetimes and did not lose their separation power following >10,000 injections. Finally, hypercrosslinked columns have been used in the 1- and 2D LC of phenolic compounds.

A novel derivatization reagent possessing a bromoquinolinium structure for biological carboxylic acids in HPLC‐ESI‐MS/MS

A novel bromoquinolinium reagent, i.e. 1-(3-aminopropyl)-3-bromoquinolinium bromide (APBQ), was synthesized for the analysis of carboxylic acids. A simple and practical precolumn derivatization procedure using the APBQ in RP chromatography and MS (HPLC-MS) has been developed using bile acids and free fatty acids, as the representative carboxylic acids in biological samples. The APBQ efficiently reacted with carboxylic acids at 60°C for 60 min in the presence of N,N-dicyclohexylcarbodiimide and pyridine as the activation reagents. Because the APBQ possesses a bromine atom in the structure, the identification of a series of carboxylic acids was easily achieved due to the characteristic bromine isotope pattern in the mass spectra. The APBQ also has a quaternary amine structure, thus the positively charged derivatives are predominate for the highly sensitive detection of carboxylic acids. The APBQ was successfully applied to the selective determination of biological carboxylic acids in human plasma. The bile acids (chenodeoxycholic acid and deoxycholic acid) and several saturated (stearic acid and palmitic acid) and unsaturated free fatty acids (oleic acid and linoleic acid) were reasonably determined by HPLC-MS under the proposed procedure. Based on the results of analyses of human plasma and saliva, the proposed procedure using APBQ seems to be applicable for the qualitative and quantitative analyses of a series of carboxylic acids in biological samples.