Cationic Ion-pairing Reagents Research Articles

Ion-Pairing with Triethylammonium Acetate Improves Solid-Phase Extraction of ADP-Ribosylated Peptides.

ADP-ribosylation refers to the post-translational modification of protein substrates with monomers or polymers of the small molecule ADP-ribose. ADP-ribosylation is enzymatically regulated and plays roles in cellular processes including DNA repair, nucleic acid metabolism, cell death, cellular stress responses, and antiviral immunity. Recent advances in the field of ADP-ribosylation have led to the development of proteomics approaches to enrich and identify endogenous ADP-ribosylated peptides by liquid chromatography tandem mass spectrometry (LC-MS/MS). A number of these methods rely on reverse-phase solid-phase extraction as a critical step in preparing cellular peptides for further enrichment steps in proteomics workflows. The anionic ion-pairing reagent trifluoroacetic acid (TFA) is typically used during reverse-phase solid-phase extraction to promote retention of tryptic peptides. Here we report that TFA and other carboxylate ion-pairing reagents are inefficient for reverse-phase solid-phase extraction of ADP-ribosylated peptides. Substitution of TFA with cationic ion-pairing reagents, such as triethylammonium acetate (TEAA), improves recovery of ADP-ribosylated peptides. We further demonstrate that substitution of TFA with TEAA in a proteomics workflow specific for identifying ADP-ribosylated peptides increases identification rates of ADP-ribosylated peptides by LC-MS/MS.

Retention of glycopeptides analyzed using hydrophilic interaction chromatography is influenced by charge and carbon chain length of ion-pairing reagent for mobile phase.

Characterization of the glycans of glycoproteins is essential for the development and production of biologics. Numerous methods are available for analyzing the glycans of glycoproteins directly and labeled glycans. Nevertheless, glycopeptides are difficult to resolve because of their exceptional complexity and the microheterogeneity of glycans. These properties represent technical challenges to efforts to insure the accurate characterization of biopharmaceuticals to comply with regulatory requirements. Therefore, we investigated the retention behavior of peptides and glycopeptides in hydrophilic interaction chromatography-mode HPLC in the presence of ion-pairing reagents. Anionic ion-pairing reagents decreased the retention times of glycopeptides and improved resolution in the presence of higher concentrations or hydrophobicities of ion-pairing reagent. Anionic ion-pairing reagents increased retention times of larger glycans because of their increased hydrophilicity. In contrast, in the presence of cationic ion-pairing reagents, the retention times of glycopeptides with greater numbers of sialic acid residues decreased. It is appropriate to add an anionic ion-pairing reagent to the mobile phase for good separation of glycopeptides. The collision cross-sectional area values of glycopeptides determined using electrospray ionization-ion mobility spectrometry-mass spectrometry correlated with retention times. These findings support the implementation of hydrophilic interaction chromatography-mode HPLC to improve the characterization of glycosylated biopharmaceuticals.

Hydrophilic interaction chromatography coupled to tandem mass spectrometry in the presence of hydrophilic ion-pairing reagents for the separation of nucleosides and nucleotide mono-, di- and triphosphates

A fast and efficient method for the simultaneous separation of highly polar compounds, in this case nucleosides and nucleotide mono-, di- and triphosphates, using hydrophilic interaction chromatography coupled with tandem mass spectrometry (HILIC–MS/MS) is proposed. This new separation method revealed the possibilities of the formation of hydrophilic ion-pairing compounds.Three stationary phases (HILIC XBridge-Amide, HILIC-CoreShell and ZIC-HILIC) were assayed for the separation of 20 target analytes, and a detailed study of the composition of the mobile phase was made using different salts at different concentrations in a organic-rich mobile phase. We report that in order to prevent the adsorption of nucleotides on the LC–MS setup and to enhance their retention on the HILIC stationary phase, a mobile phase containing hexafluoro-2-propanol and different cations should be used. Four cations were evaluated: ammonium, diethylammonium, triethylammonium and tetrabutylammonium. The results revealed the formation of an ionic-association compound between the phosphorylated analytes and the cationic ion-pairing reagents, whose retention increased with the polarity of the cationic ion-pairing reagent.HILIC XBridge-Amide was found to be the most suitable column for the separation of these analytes, and the optimized mobile phase consisted of an ACN/UHQ water mixture (3min of isocratic elution using 82:18%, v/v and then a fast gradient from 18% to 22% of water) with 100mM hexafluoro-2-propanol and 50mM diethylamine (wwpH 9–wspH 10). In a total analysis time of 8min, good results were achieved in terms of resolution. Under these optimum conditions, a further comprehensive study of the retention mechanism was carried out.

Optimized chromatographic conditions for separation of halogenated acetic acids by ultra-performance liquid chromatography–electrospray ionization-mass spectrometry

Emerging halogenated acetic acids (HAAs), especially mixed halogenated acids such as chlorobromo-, chloroiodo- and bromoiodo-acetic acids, are unregulated disinfection by-products in drinking water. Because these compounds are hydrophilic and strongly acidic, they are difficult to detect at trace levels using approved analytical methods. In the present study, 13 HAAs were effectively separated on three ultra-performance liquid chromatography columns. The effects of changing in the aqueous mobile phase, acidic solutions and cationic volatile ion pair reagents were investigated. The samples were pretreated by filtration, and extraction, while derivatization and concentration procedures were not required. The limits of quantitation for regulated HAAs were between 0.5μg/L and 1.7μg/L and for unregulated HAAs were 1.2 and 5.8μg/L, especially for the iodinated acetic acids were 1.5 and 2.1μg/L. The method was applied to two finished water samples collected in China (Shanghai and Xuzhou) from water treatment plants that use chlorine for disinfection. Multiple unregulated HAAs were found in the two samples, but iodoacids were only detected in the water sample from Shanghai, which could be attributed to the characteristics of the source water. The presence of unregulated HAAs, especially mixed bromo- and iodoacetic acids, in the finished water samples could affect human health, and this warrants further investigation.

Sensitive analysis of metal cations in positive ion mode electrospray ionization mass spectrometry using commercial chelating agents and cationic ion-pairing reagents

Metals play a very important role in many scientific and environmental fields, and thus their detection and analysis is of great necessity. A simple and very sensitive method has been developed herein for the detection of metals in positive ion mode ESI-MS. Metal ions are positively charged, and as such they can potentially be detected in positive ion mode ESI-MS; however, their small mass-to-charge (m/z) ratio makes them fall in the low-mass region of the mass spectrum, which has the largest background noise. Therefore, their detection can become extremely difficult. A better and well-known way to detect metals by ESI-MS is by chelating them with complexation agents. In this study eleven different metals, Fe(II), Fe(III), Mg(II), Cu(II), Ru(III), Co(II), Ca(II), Ni(II), Mn(II), Sn(II), and Ag(I), were paired with two commercially available chelating agents: ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS). Since negative ion mode ESI-MS has many disadvantages compared to positive ion mode ESI-MS, it would be very beneficial if these negatively charged complex ions could be detected in the positive mode. Such a method is described in this paper and it is shown to achieve much lower sensitivities. Each of the negatively charged metal complexes is paired with six cationic ion-pairing reagents. The new positively charged ternary complexes are then analyzed by ESI-MS in the positive single ion monitoring (SIM) and single reaction monitoring (SRM) modes. The results clearly revealed that the presence of the cationic reagents significantly improved the sensitivity for these analytes, often by several orders of magnitude. This novel method developed herein for the detection of metals improved the limits of detection (LODs) significantly when compared to negative ion mode ESI-MS and shows great potential in future trace studies of these and many other species.

Use of ion pairing reagents for sensitive detection and separation of phospholipids in the positive ion mode LC-ESI-MS

Phospholipids make up one of the more important classes of biological molecules. Because of their amphipathic nature and their charge state (e.g., negatively charged or zwitterionic) detection of trace levels of these compounds can be problematic. Electrospray ionization mass spectrometry (ESI-MS) is used in this study to detect very small amounts of these analytes by using the positive ion mode and pairing them with fifteen different cationic ion pairing reagents. The phospholipids used in this analysis were phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), cardiolipin (CA) and sphingosyl phosphoethanolamine (SPE). The analysis of these molecules was carried out in the single ion monitoring (SIM) positive mode. In addition to their detection, a high performance liquid chromatography and mass spectrometry (HPLC-MS) method was developed in which the phospholipids were separated and detected simultaneously within a very short period of time. Separation of phospholipids was developed in the reverse phase mode and in the hydrophilic interaction liquid chromatography (HILIC) mode HPLC. Their differences and impact on the sensitivity of the analytes are compared and discussed further in the paper. With this technique, limits of detection (LODs) were very easily recorded at low ppt (ng L(-1)) levels with many of the cationic ion pairing reagents used in this study.

Detection of nucleotides in positive-mode electrospray ionization mass spectrometry using multiply-charged cationic ion-pairing reagents

Nucleotides are a class of molecules that play an essential role in biological systems. A new method has been developed in the detection of nucleotides. These molecules can exist as monomers or constituents of oligomers and polymers. As such, they carry from one to several negative charges. In this study, different cationic ion-pairing reagents were used to complex with each of the 28 nucleotide monomers and nucleotide containing compounds. By using this method, this discrete set of anions was able to be detected in the positive-mode electrospray ionization mass spectrometry, as positively charged complexes. Tandem mass spectrometry experiments were also completed on the ion-pairing reagents that performed the best in the single ion monitoring (SIM) ion mode, and the sensitivity was lowered even further for most of the anions. Limits of detection for compounds such as thymidine diphosphate were improved as much as 100 times compared to the positive SIM mode, and 750 times when compared to the negative mode. A few nucleotides did not show a significant increase in sensitivity when analyzed in the positive ion mode, but in general the new method developed herein resulted in a much greater sensitivity than traditional detection in the negative mode.

Replacement of acetonitrile by ethanol as solvent in reversed phase chromatography of biomolecules

Acetonitrile, which is a by-product of acrylonitrile synthesis, is the commonly used solvent in ion-pair reversed phase chromatography. In consequence of the decreasing demand for acrylonitrile due to the financial crisis, a worldwide shortage of acetonitrile is observed. Therefore, the aim of this study was to establish ion-pair reversed phase chromatographic assays using alternative eluents for acetonitrile and to decrease costs incurred hereby. We compared the performance of ion-pair reversed phase chromatography using acetonitrile with the alternative eluents methanol, ethanol and n-propanol, using monolithic reversed phase C5 as well as C18 chromatography columns. We used triethylammonium acetate (TEAA) and tetrabutylammonium sulfate (TBA) as representative cationic ion-pair reagents and trifluoroacetic acid (TFA) as representative anionic ion-pair reagent. For covering a large field of applications, we fractionated representative low, middle and high-molecular weight biomolecules, in particular dinucleoside polyphosphates, peptides, proteins and tryptic digested human serum albumin. Whereas the chromatographic characteristics of both methanol and n-propanol were partly insufficient, ethanol was characterised equally or partly even better in the matter of elution strength and separation quality compared to the eluent water–acetonitrile. In conclusion, ethanol is an appropriate alternative for acetonitrile in ion-pair reversed phase chromatography of biomolecules.

Separation of a range of cations by nonaqueous capillary electrophoresis using indirect and direct detection

The use of nonaqueous media and indirect detection is reported for the separation and detection of a range of small cations. The novel applications involved separation of a range of metal ions, small nonchromophoric amines, cationic ion-pair reagents and cationic surfactants. Separations were achieved using acidified methanol containing imidazole as the UV co-ion for indirect detection. The methods produced different selectivity compared to aqueous methods using acidified aqueous imidazole solutions. Advantages of the methods include speed of analysis and prevention of sample micellerisation. The methods were shown to be quantitative and reproducible by their application to the determination of Tris content.

Distribution of cationic ion-pairing reagents on thin-layers after continuous overpressured layer chromatography

Ion-pair HPLC separations on layers were modelled by using continuous overpressured layer chromatography (OPLC). As a first step, the distribution of several cationic ion-pairing reagents on the layer was studied. The mobile phase containing different quaternary ammonium salts, such as tetramethylammonium (TMA), tetrabutylammonium (TBA), cetyltrimethylammonium (CTMA) and trioctylmethylammonium (TOMA) halides was continuously pumped through the stationary phase at a pressure of 40 bar. The volume of the mobile phase varied between 4.5 to 67.5 cm 3 which is equivalent to a 1 to 15 bed volume of the given silica-gel layer. The concentration of the reagents was measured after chromatography in ten samples of the stationary phase taken along the direction of the development. Silica F-gel and physically and chemically bonded reversed-phase layers (C 18) were used. In some cases the effluents also were analyzed. With 0.05 M CTMA in 50% methanol–water mixture, the layers acted as `spread out columns' and they could be conditioned after pumping through 22.5 cm 3 mobile phase. This means that there was no concentration gradient on the layer and the remaining reagent quantities on the layer did not change with increasing volumes of the mobile phase. In some cases, the behaviour of TMA was different from that of CTMA.

A systematic study of the desorption of different ion-pairing reagents on reversed phase thin-layers

The absolute concentrations and the concentration distribution of different cationic ion-pairing reagents, such as tetramethylammonium bromide, tetrabutylammonium bromide, cetyltrimethylammonium bromide and trioctylmethylammonium chloride were studied after chromatographic development on physically and chemically bonded reversed-phase layers. The combination of the chromatographic conditions involved three variations. Untreated layers were chromatographed with mobile phases containing the ion-pairing reagent. Several layers were treated with the ion-pairing reagent prior to the chromatographic run, and methanol-water mixtures were used as mobile phase. In a third set of experiments both the layer and the mobile phase contained the ion-pairing reagent. The chromatographic behaviour of acid, neutral and basic model compounds were also studied. For the determination of different reagents remaining on the layer after the chromatographic run, spectrophotometric, potentiometric methods and capillary electrophoresis were used. The performance of the analytical methods was evaluated. Results obtained for the absolute concentrations and distribution of the reagents and the retention data were compared to those obtained on silica gel layers. A discussion of the retention mechanism is given.

Development of an isocratic high-performance liquid chromatographic method for monitoring of ciprofloxacin photodegradation

Abstract An isocratic reversed-phase ion-pair high-performance liquid chromatographic method was developed for monitoring the photodegradation of ciprofloxacin in aqueous solutions. Changes in the chromatographic behaviour of ciprofloxacin and its degradation products under the influence of organic modifiers, anionic and cationic ion-pair reagents and pH were investigated. Baseline separation was achieved with use of an eluent of acetonitrile-phosphoric acid (20 mM, pH 2.3) containing 2.5 mM 1-heptanesulphonic acid sodium salt. The method was applied to the quantification of ciprofloxcin in irradiated solutions. The calibration graphs were linear from 5 to 150 μg/ml (r > 0.9999). Within the same range, the accuracy was 99–102% of the expected values. The intra-assay repeatability of the peak areas and retention times was good (R.S.D. ⩽ 039%). Likewise the intra-day reproducibility of the method was good: R.S.D. values (n = 6) were ⩽ 2.3% and 1.8% for the lowest and highest concentrations, respectively. The inter-day precision gave a mean R.S.D. of 2.4% for peak areas and R.S.D. of 3.3% for retention times during a working period of three months.

Study of the influence of aqueous micellar systems on the derivatization of undecylenic acid with 4-bromomethyl-7-methoxycoumarin

The effects of aqueous micellar systems on the derivatization of a fatty acid, 10-undecenoic acid (UA), with a flourophore, 4-bromomethyl-7-methoxycoumarin (BrMMC), were examined. High derivatization rates were obtained in solutions of the non-ionic surfactants Triton-X 100 and Arkopal N in the presence of cationic ion-pair reagents such as tetrahexylammonium bromide. The derivatization mechanism is probably based on phase-transfer catalysis. Especially high reaction rates are obtained in turbid non-ionic micellar solutions. This opaqueness is connected with an important optimizing parameter of the derivatization rate, the so-called cloud temperature of a micellar system. Under the optimal conditions the derivatizaton of UA with BrMMC is complete within 45 min at 70°C.

Spectral properties of some ion-pairing reagents commonly used in reversed-phase high-performance liquid chromatography of proteins and peptides in acetonitrile gradient systems

Electronic spectra of some anionic anc cationic ion-pairing reagents in aqueous solution and in solvent mixtures with various contents of acetonitrile were recorded between 200 and 300 nm, and optimum conditions for highly sensitive detection using linear solvent gradients in reversed-phase high-performance liquid chromatography were proposed. Butane-, hexane and octanesulphonic acids showed no remarkable absorbance and recorded baseline drifts are therefore mainly caused by the increasing contents of acetonitrile. For trifuloroacetic acid (TFA) and heptafluorobutyric acid (HFB), the baseline trace of a linear acetonitrile gradient system is the result of a discontinuous change from the π → π* band of the anion in aqueous solution to the n → π* transition of the undissociated acid in the organic solvent and therefore depends strongly on the wavelength used. For both acids, there exists an optimal recording point below 220 nm (TFA: 215 nm, HFB: 219 nm). Cationic hydrophobic ion-pairing reagents such as alkylated amines are weakly protonated at a higher pH and show relatively strong absorbance below 250 nm. The ionization is further reduced by the addition of acetonitrile, leading to an extreme baseline drift. At low pH, however, complete ionization is achieved even at high contents of organic solvent, but ion-pairing with the carboxylic group of peptides is then impossible. In contrast to the amines, the tetraalkylammonium cation is a relatively strong Lewis acid and the pH dependency as well as the organic solvent dependency of the electronic spectra is therefore less marked. Nevertheless, in the case of tetrabutylammonium phosphate the baseline drift shows a minimum at pH 5 and therefore this pH is highly recommended for sensitive detection using acetonitrile gradients.