Ion-pair Conditions Research Articles

Identification of horn-derived animal medicines based on detection of keratin-derived characteristic peptides

Due to the highly stable structure of keratin, the extraction and dissolution steps of animal medicines rich in keratin are complex, which seriously restricts the detection efficiency and flux. Therefore, this study simplified the pre-treatment steps of horn samples and optimized the detection methods of characteristic peptides to improve the efficiency of identifying the specificity of horn-derived animal medicines. For detection of the characteristic peptides in horn-derived animal medicines treated with/without iodoace-tamide(IAA), the ion pair conditions of the characteristic peptides were optimized, and the retention time, intensity and other data of the specific peptides were compared between the samples treated with/without IAA. Two pre-treatment methods, direct enzymatic hydrolysis and total protein extraction followed by enzymatic hydrolysis, were used to prepare horn-derived animal medicine samples. The effects of different methods on the detection of specific peptides in the samples of Saiga antelope horn, water buffalo horn, goat horn, and yak horn were compared regarding the retention time of specific peptides and ion intensity. The results indicated that after direct enzymatic hydrolysis, the specific peptides in the samples without IAA treatment can be detected. Compared with the characteristic peptides in the samples treated with IAA, their retention time shifted back and the mass spectrometry response slightly decreased. The specific peptides of the samples without IAA treatment had good specificity and did not affect the specificity identification of horn-derived animal medicines. Overall, the process of direct enzymatic hydrolysis can be used to treat horn samples, omitting the steps of protein extraction and dithiothreitol and IAA treatment, significantly improving the pre-treatment efficiency without affecting the specificity identification of horn-derived animal medicines. This study provides ideas for quality research and standard improvement of horn-derived animal medicines.

Investigation of factors influencing the separation of diastereomers of phosphorothioated oligonucleotides

This study presents a systematic investigation of factors influencing the chromatographic separation of diastereomers of phosphorothioated pentameric oligonucleotides as model solutes. Separation was carried out under ion-pairing conditions using an XBridge C18 column. For oligonucleotides with a single sulfur substitution, the diastereomer selectivity was found to increase with decreasing carbon chain length of the tertiary alkylamine used as an ion-pair reagent. Using an ion-pair reagent with high selectivity for diastereomers, triethylammonium, it was found the selectivity increased with decreased ion-pair concentration and shallower gradient slope. Selectivity was also demonstrated to be dependent on the position of the modified linkage. Substitutions at the center of the pentamer resulted in higher diastereomer selectivity compared to substitutions at either end. For mono-substituted oligonucleotides, the retention order and stereo configuration were consistently found to be correlated, with Rp followed by Sp, regardless of which linkage was modified. The type of nucleobase greatly affects the observed selectivity. A pentamer of cytosine has about twice the diastereomer selectivity of that of thymine. When investigating the retention of various oligonucleotides eluted using tributylammonium as the ion-pairing reagent, no diastereomer selectivity could be observed. However, retention was found to be dependent on both the degree and position of sulfur substitution as well as on the nucleobase. When analyzing fractions collected in the front and tail of overloaded injections, a significant difference was found in the ratio between Rp and Sp diastereomers, indicating that the peak broadening observed when using tributylammonium could be explained by partial diastereomer separation.

Bicyclo[6.3.0]undecapentaenyl Anion: The Next Higher Homolog of the Indenyl Anion with Exceptionally Large Ion-Pairing Effects on its Tropicity

The title anion 1 was generated as a fairly thermally stable species in tetrahydrofuran (THF) and dimethylsulfoxide (DMSO) by the action of several bases (sodium hydride, potassium hydride, lithium diisopropylamide, and lithium hexamethyldisilazide) with appropriate bicyclo[6.3.0]undecapentaenes. Variable-temperature 1H NMR spectra of 1⋅Li+ in [D8]THF reveal that the anion exhibits exceptionally large ion-pairing effects; proton chemical shifts vary by more than 1 ppm as a function of ion-pairing conditions. Thus, anion 1, in a contact ion pair (Li+ at ambient temperature in THF), behaves as an aromatic cyclopentadienyl anion that is perturbed only slightly by the electronic effects of a paramagnetic cyclooctatetraene (COT), whereas 1 in a separated ion pair (Li+ at low temperatures in THF or at ambient temperature in DMSO) behaves as an overall paratropic species with a 12 π-electron periphery. 13C NMR spectroscopy indicates no major skeletal rearrangement and only small variations of the electron density. The variable tropicity of 1 can be ascribed to small conformational changes of the molecule. In addition to its unusual, tunable tropicity, anion 1 can also serve as a versatile building block for the synthesis of cyclopentanoid conjugated systems fused to a fully unsaturated eight-membered ring. A theoretical calculation predicts that the 10-position of 1 should have the highest electron density. In agreement with this prediction, the reactions of 1 with electrophiles occur predominantly at the 10-position. The corresponding ferrocene, two fulvenes, two diazo derivatives, and a COT-fused azulene were obtained by the reactions of 1 with appropriate electrophiles.

Heparin‐like glycosaminoglycan/amine salt‐bridge interactions: A new potential tool for HLGAGs analysis using mass spectrometry

Characterization of glycosaminoglycans poses a challenge for current analytical techniques, as they are highly acidic, polydisperse and heterogeneous compounds. The purpose of this study is the separation and analysis of a partially depolymerized heparin-like glycosaminoglycan by on-line ion-pairing reversed-phase high-performance liquid chromatography/electrospray mass spectrometry. The gas-phase behavior of two synthesized glycosaminoglycans has been investigated. Dibutylamine was found to be the best suited ion-pairing reagents for mass spectrometry analysis. The optimized ion-pairing conditions provide reproducible and easily interpretable electrospray mass spectra in both negative and positive ESI modes. The glycosaminoglycans are detected as a non-covalent complex with amines. In fact, the observed ionic species and their gas-phase dissociation under CID conditions revealed the presence of salt bridge interactions in the gas phase.

Characterization of LipL as a Non-heme, Fe(II)-dependent α-Ketoglutarate:UMP Dioxygenase That Generates Uridine-5′-aldehyde during A-90289 Biosynthesis

Fe(II)- and α-ketoglutarate (α-KG)-dependent dioxygenases are a large and diverse superfamily of mononuclear, non-heme enzymes that perform a variety of oxidative transformations typically coupling oxidative decarboxylation of α-KG with hydroxylation of a prime substrate. The biosynthetic gene clusters for several nucleoside antibiotics that contain a modified uridine component, including the lipopeptidyl nucleoside A-90289 from Streptomyces sp. SANK 60405, have recently been reported, revealing a shared open reading frame with sequence similarity to proteins annotated as α-KG:taurine dioxygenases (TauD), a well characterized member of this dioxygenase superfamily. We now provide in vitro data to support the functional assignment of LipL, the putative TauD enzyme from the A-90289 gene cluster, as a non-heme, Fe(II)-dependent α-KG:UMP dioxygenase that produces uridine-5'-aldehyde to initiate the biosynthesis of the modified uridine component of A-90289. The activity of LipL is shown to be dependent on Fe(II), α-KG, and O(2), stimulated by ascorbic acid, and inhibited by several divalent metals. In the absence of the prime substrate UMP, LipL is able to catalyze oxidative decarboxylation of α-KG, although at a significantly reduced rate. The steady-state kinetic parameters using optimized conditions were determined to be K(m)(α-KG) = 7.5 μM, K(m)(UMP) = 14 μM, and k(cat) ≈ 80 min(-1). The discovery of this new activity not only sets the stage to explore the mechanism of LipL and related dioxygenases further but also has critical implications for delineating the biosynthetic pathway of several related nucleoside antibiotics.

Retention Behaviour of Bupropion Hydrochloride in Reversed Phase Ion Pair LC and Validated Analysis of the Drug in Pharmaceuticals

The chromatographic behaviour of bupropion hydrochloride, a basic drug of pKa 7.9, has been investigated under reversed-phase ion-pairing conditions and the results were used to develop a method for analysis of bupropion hydrochloride in pharmaceuticals. Chromatographic separation of bupropion hydrochloride and carbamazepine (used as internal standard) was performed on a C8 column (150 mm × 4.6 mm i.d., 3.5-μm particle), with 40:10:50 (v/v) methanol–acetonitrile–phosphate buffer (20 mm, pH 3.0), containing 10 mm 1-heptane sulfonic acid sodium salt (1-HSA), as optimum mobile phase at a flow rate of 1.0 mL min−1. UV detection was at 254 nm. The fully validated method enables reproducible and selective analysis of bupropion hydrochloride in pharmaceuticals.

Stereoselectivity in Ene Reactions with 1O2: Matrix Effects in Polymer Supports, Photo‐oxygenation of Organic Salts and Asymmetric Synthesis

The ene reaction of chiral allylic alcohols is applied as a tool for the investigation of intrapolymer effects by means of the stereoselectivity of the singlet-oxygen addition. The diastereo selectivity strongly depends on the structure of the polymer, the substrate loading degree and also on the degree of conversion demonstrating additional supramolecular effects evolving during the reaction. The efficiency and the stability of polymer-bound sensitizers were evaluated by the ene reaction of singlet oxygen with citronellol. The ene reaction with chiral ammonium salts of tiglic acid was conducted under solution phase conditions or in polystyrene beads under chiral contact ion-pair conditions. The products thus obtained precipitate during the photoreaction as ammonium salts. Moderate asymmetric induction was observed for this procedure for the first time.

Prediction of internal standards in reversed-phase liquid chromatography: IV. Correlation and prediction of retention in reversed-phase ion-pair chromatography based on linear solvation energy relationships

This paper describes the results of the evaluation of a new solvation parameter model for reversed-phase ion-pair chromatography by linear gradient elution. This model is described as t = Int + r ′ R + s ′ π 2 H + a ′ ∑ α 2 H + b ′ ∑ β 2 H + m ′ V X + U I / ( 1 + V × 10 ± ( p K a − pH ) ) + γ IP Z S . The first six terms are the usual solvation parameter equation for neutral solutes, and the seventh term represents the contribution to retention from solute’s ionization. The last term describes the retention increase due to ion-pair effect. Retention times obtained for 60 solutes (neutral, acidic and basic) in acetonitrile/aqueous mobile phases with different ion-pair reagents (phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, perchloric acid, and hexafluorophosphoric acid) are used to evaluate the capability of the function. It is concluded that the model describes the retention of ionizable/ionized compounds under ion-pair conditions very well. Accordingly, the function extends the application of linear solvation energy relationships (LSERs) to ionizable compounds in ion-pair chromatography, and allows us to easily predict their retention for chromatographic optimization, including selectivity optimization and internal standard selection. Finally, the conclusion can be extended to ioscratic elution.

Prediction of internal standards in reversed-phase liquid chromatographyIV. Correlation and prediction of retention in reversed-phase ion-pair chromatography based on linear solvation energy relationships

Abstract This paper describes the results of the evaluation of a new solvation parameter model for reversed-phase ion-pair chromatography by linear gradient elution. This model is described as t = Int + r ′ R + s ′ π 2 H + a ′ ∑ α 2 H + b ′ ∑ β 2 H + m ′ V X + U I / ( 1 + V × 10 ± ( p K a − pH ) ) + γ IP Z S . The first six terms are the usual solvation parameter equation for neutral solutes, and the seventh term represents the contribution to retention from solute’s ionization. The last term describes the retention increase due to ion-pair effect. Retention times obtained for 60 solutes (neutral, acidic and basic) in acetonitrile/aqueous mobile phases with different ion-pair reagents (phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, perchloric acid, and hexafluorophosphoric acid) are used to evaluate the capability of the function. It is concluded that the model describes the retention of ionizable/ionized compounds under ion-pair conditions very well. Accordingly, the function extends the application of linear solvation energy relationships (LSERs) to ionizable compounds in ion-pair chromatography, and allows us to easily predict their retention for chromatographic optimization, including selectivity optimization and internal standard selection. Finally, the conclusion can be extended to ioscratic elution.

Optimization study for the reversed-phase ion-pair liquid chromatographic determination of nicotine in commercial tobacco products

The availability of published methods for the determination of nicotine in commercial tobacco products based on state-of-the-art chromatographic methods is limited. Nicotine is a diprotic base with p K a’s of 3.12 (pyridine ring) and 8.02 (pyrrolidine ring). Other monoprotic and diprotic bases are also present in commercial tobacco including anatabine, nornicotine, anabasine, and cotinine. In this paper, the chromatography of nicotine and the minor tobacco alkaloids under reversed-phase ion-pairing conditions is thoroughly studied. The results of this study are used to understand the retention mechanisms of the tobacco alkaloids, to examine their observed elution order with respect to fundamental analyte properties (size, functionality, and acid–base strength), and to select optimum chromatographic conditions for the determination of nicotine in commercial tobacco products.

Analysis of cyclic nucleotides and cytokinins in minute plant samples using phase-system switching capillary electrospray-liquid chromatography-tandem mass spectrometry

Using an electrospray tandem mass spectrometer as a concentration-sensitive detector, a method has been developed to quantify femtomole amounts of plant growth regulators (i.e. isoprenoid type cytokinins, zeatin, dihydrozeatin, isopentenyladenine and their respective riboside and glucoside analogues) and the second messenger adenosine 3′:5′-cyclic monophosphate (3′:5′-cAMP). Miniaturisation of the chromatographic setup using capillary high performance liquid chromatographic (HPLC) ion spray mass spectrometry increased the sensitivity to the low femtomole region. Application of automated capillary column switching allowed the introduction of large injection volumes into the HPLC system. Aliquots (25 µL) were injected into one dimension of the HPLC set-up and stacked onto a micro pre-column. By means of mobile phase switching the pre-column was back-flushed to introduce the analytes onto the analytical column. For cytokinin analysis positive electrospray ionisation was used and resulted in 2.5–25 fmol detection limits. Cyclic nucleotides were separated under ion-pair conditions using tetrabutyl ammonium bromide as ion-pair reagent and were detected under negative electrospray ionisation conditions. Here a 25 fmol detection limit was determined. Following this approach, cytokinins and 3′:5′-cAMP extracted from only mg amounts of apical shoot meristem and chloroplasts obtained from Nicotiana tabacum cv. Petit Havana SR1 were identified and quantified. Copyright © 1999 John Wiley & Sons, Ltd.

Quantitative Structure Retention Relationship Studies of Some Basic Antimalarial Compounds

Abstract Studies have been made of the correlation between chromatographic retention parameter, k' and solute hydrophobicity as determined by the oil-in-water partition coefficient. Using a set of 29 basic antimalarial drugs and analogues such correlations have been made under ion pairing and non ion pairing conditions. The capacity factors have been obtained extrapolated to pure water and in the case of ion pairing conditions, the k'max values have been used. It is shown that better correlations can be obtained under the ion pairing conditions. The use of oppositely charged ion pairing agents can provide an alternative to the inclusion of hydrophobic alkylamines in order to overcome putative effects of residual silanols.

RP-HPLC Separation of Highly Charged Quaternary Ammonium Salts

Abstract The reverse phase HPLC separation of polycationic viologenes 1 – 4 and macrotricyclic quaternary ammonium salts 5 – 8 using ion pair conditions is described. Whereas the compounds 1 – 4 could be analysed on any of 4 stationary RP-18 phases tested, the cage compounds 5 – 8 were much more sensitive to the source of the matrix material. Optimal separation conditions for the latter up to eightfold positively charged macrocycles employ sodium perchlorate in acidic aqueous methanol using Nucleosil RP-18 or Lichrospher CH-18 columns.

A carbon‐13 NMR study of the ion pair structure of the dibenzo[b,f]pentalene dianion

AbstractThe 1H and 13C NMR chemical shifts of dibenzo[b, f]pentalene and its 5,10‐dimethyl derivative are presented and compared with those of the corresponding dilithium dianions. As probed by the relative 13C NMR chemical shifts, the charge distribution within the dianion system is clearly dependent on the actual ion pair state. This condition is demonstrated by varying the solvent and temperature. The polarization of charge towards the pentalene carbons, i.e. the preferred cation positions, is observed on going to tight ion pair conditions. Further support for this model is gained from 7Li NMR. The limitations of the use of 1H and 13C NMR chemical shifts to measure charge distributions within anion systems are discussed.

The determination of d-penicillamine and its disulphide in plasma by reversed-phase ion-pair high-performance liquid chromatography

Reversed-phase ion-pair conditions are used for the determination of D-penicillamine and penicillamine disulphide. Two chromatographic systems were employed, one for penicillamine and the other for penicillamine disulphide. The procedures permit the determination of total penicillamine (protein-bound, free and as disulphides) in whole plasma, and total penicillamine (free and as disulphides) in plasma ultrafiltrate, using an incubation step in the presence of dithiothreitol. Free penicillamine and penicillamine disulphide may be determined independently by direct injection of plasma ultrafiltrate. Both solutes may be measured at an on-column sensitivity of 10 ng, utilizing an electrochemical detector based on a glassy carbon electrode.

A general occurence of the syn-anti elimination dichotomy under ion-pairing conditions : inessentiality of steric requirements of the leaving group

A general occurence of the syn-anti elimination dichotomy under ion-pairing conditions : inessentiality of steric requirements of the leaving group