Determination of antazoline hydrochloride in rat plasma and excreta by reversed‐phase ion‐pair chromatography and its application to pharmacokinetics

A reversed‐phase ion pair chromatography method with liquid–liquid extraction analytical method was developed and validated for the determination of antazoline hydrochloride in plasma and excreta of rat. The aim of our study was to characterize the preclinical pharmacokinetics and excretion profiles of antazoline hydrochloride in rats after intravenous injection at the dose of 10 mg/kg. Plasma and excreta samples were extracted with ethyl acetate, and phenacetin was used as the internal standard. The result showed that the method is suitable for the quantification of antazoline hydrochloride in plasma and excreta samples. Analysis of accuracy (90.89–112.33%), imprecision (<7.1%) and recovery (>82.5%) showed adequate values. After a single intravenous administration at 10 mg/kg to rats, plasma concentration profile showed a relative fast elimination proceeding with a terminal elimination half‐life of 3.53 h. Approximately 61.8 and 14.2% of the administered dose were recovered in urine and bile after 72 and 24 h post‐dosing respectively; 5.9% of the administered dose was recovered in feces after 72 h post‐dosing. The above results show that the major elimination route is urinary excretion. Copyright © 2013 John Wiley & Sons, Ltd.     

Reversed‐phase ion‐pair solid‐phase extraction and ion chromatography analysis of pyrrolidinium ionic liquid cations in environmental water samples

A method of reversed‐phase ion‐pair solid‐phase extraction combined with ion chromatography for determination of pyrrolidinium ionic liquid cations (N‐methyl‐N‐ethyl pyrrolidinium, N‐methyl‐N‐propyl pyrrolidinium, and N‐methyl‐N‐butyl pyrrolidinium) in water samples was developed in this study. First, ion‐pair reagent sodium heptanesulfonate was added to the water samples after static, centrifugation and filteration. Then, pyrrolidinium cations in the samples were enriched and purified by a reversed‐phase solid‐phase extraction column, and eluted from the column with methanol aqueous solution as eluent. Finally, the eluate collected was analyzed by ion chromatography. The separation and direct conductivity detection of these pyrrolidinium cations by ion‐exchange column using 1.0 mM methanesulfonic acid (in water)/acetonitrile (97:3, v:v) as mobile phase was achieved within 10 min. By using this method, pyrrolidinium cations in Songhua River and Hulan River were successfully extracted with the recoveries ranging from 74.2 to 97.1% and the enrichment factor assessed as 60. Pyrrolidinium cations with the concentration of 0.001?0.03 mg/L can be enriched and detected in the water samples. The developed method for the determination of pyrrolidinium ionic liquid cations in water samples is simple and reliable, which provides a reference for the study of the potential impact of ionic liquids on the environment.     

Retention of nucleic acids in ion‐pair reversed‐phase high‐performance liquid chromatography depends not only on base composition but also on base sequence

The study on nucleic acid retention in ion‐pair reversed‐phase high‐performance liquid chromatography mainly focuses on size‐dependence, however, other factors influencing retention behaviors have not been comprehensively clarified up to date. In this present work, the retention behaviors of oligonucleotides and double‐stranded DNAs were investigated on silica‐based C₁₈ stationary phase by ion‐pair reversed‐phase high‐performance liquid chromatography. It is found that the retention of oligonucleotides was influenced by base composition and base sequence as well as size, and oligonucleotides prone to self‐dimerization have weaker retention than those not prone to self‐dimerization but with the same base composition. However, homo‐oligonucleotides are suitable for the size‐dependent separation as a special case of oligonucleotides. For double‐stranded DNAs, the retention is also influenced by base composition and base sequence, as well as size. This may be attributed to the interaction of exposed bases in major or minor grooves with the hydrophobic alky chains of stationary phase. In addition, no specific influence of guanine and cytosine content was confirmed on retention of double‐stranded DNAs. Notably, the space effect resulted from the stereostructure of nucleic acids also influences the retention behavior in ion‐pair reversed‐phase high‐performance liquid chromatography.     

Reversed‐phase ion‐pair high‐performance liquid chromatography assay of polyprenyl diphosphate oligomer homologues

A reversed‐phase ion‐pair high‐performance liquid chromatography procedure was developed for the separation of polyprenyl diphosphate oligomer homologues obtained chemically from plant polyprenols. Tetrabutylammonium phosphate was used as the ion‐pair reagent, and the dependence of the separation quality on pH of ion‐pair reagent was investigated for the first time. The procedure is applicable for the control of commercial available polyprenyl monophosphates (the active components of veterinary drugs Phosprenyl and Gamapren) for the possible presence of polyprenyl diphosphate byproducts.     

Separation of selenium species and their sensitive determination in rice samples by ion‐pairing reversed‐phase liquid chromatography with inductively coupled plasma tandem mass spectrometry

A highly sensitive method was developed for the simultaneous separation and determination of organic and inorganic selenium species in rice by ion‐pairing reversed‐phase chromatography combined with inductively coupled plasma tandem mass spectrometry. To achieve a good separation of these species, a comparison between anion‐exchange chromatography and ion‐pairing reversed‐phase chromatography was performed. The results indicated that ion‐pairing reversed‐phase chromatography was more suitable due to better separation and higher sensitivity for all analytes. In this case, a StableBond C18 column proved to be more robust or to have a better resolution than other C18 columns, when 0.5 mM tetrabutylammonium hydroxide and 10 mM ammonium acetate at pH 5.5 were used as the mobile phase. Moreover, an excellent sensitivity was obtained in terms of interferences by means of tandem mass spectrometry in the hydrogen mode. The detection limits were 0.02–0.12 μg/L, and recoveries of five selenium species were 75–114%, with relative standard deviations ≤ 9.4%. This method was successfully applied to the analysis of rice samples. Compared with previous studies, the proposed method not only gave comparable results when used for measuring selenium‐enriched rice, but it can provide greater sensitivity for the detection of low concentrations of selenium species in rice.     

Structural modifications of dicationic acetylcholinesterase reactivators studied under ion‐pairing mechanism in reversed‐phase liquid chromatography

A study focused on the chromatographic behavior of several acetylcholinesterase reactivators under ion‐pairing mechanism is reported. Among these reactivators, dicationic oximes and carbamoyl‐based pyridinium congeners were studied, which form ion pairs with alkylsulfonate anions. This mechanism was studied for some major experimental parameters, such as the chain length of the ion‐pairing agent added to the aqueous phase, its concentration, temperature, and nature of the organic modifier from mobile phase. Retention data showed one or two possibilities of forming ion pairs and the tautomerism of the studied reactivators, for different pH values of the aqueous component. Double sigmoid shapes were obtained for the studied compounds for the dependence between retention factor and pH, indicating the possibility of one or two tautomeric equilibria: at pH close to 7 these compounds are not stable as dicationic species and they participate in the retention process as nitroso forms, which are not able to form ion pairs with alkylsulfonates. The dependences of the retention factor on the organic modifier content from mobile phase were linear. Two complementary theoretical models were used to explain the functional dependences for the retention data on the experimental parameters.     

Method development for separating organic carbonates by ion‐moderated high‐performance liquid chromatography

An ion‐moderated partition high‐performance liquid chromatography method was developed for the separation and identification of common organic carbonates. The separation of organic carbonates was achieved on an ion exclusion column with an exchangeable hydrogen ion. An isocratic, aqueous mobile phase was used for elution and detection was performed with a refractive index detector. The developed method was validated for specificity, linearity, limits of detection and quantification, precision and accuracy. All calibration curves showed excellent linear regression (R² > 0.9990) within the testing range. The limits of detection were 3.8–30.8 ppm for the analyzed carbonates. Improvements in the peak resolution of the chromatograms were achieved by decreasing the column temperature. Addition of the organic modifier, acetonitrile, to the eluent was found to have insignificant effects on the peak resolution. The developed method was demonstrated for analyzing organic carbonate components in the electrolyte system of a commercial lithium ion battery.     

Dicationic imidazolium ionic liquid modified silica as a novel reversed‐phase/anion‐exchange mixed‐mode stationary phase for high‐performance liquid chromatography

A dicationic imidazolium ionic liquid modified silica stationary phase was prepared and evaluated by reversed‐phase/anion‐exchange mixed‐mode chromatography. Model compounds (polycyclic aromatic hydrocarbons and anilines) were separated well on the column by reversed‐phase chromatography; inorganic anions (bromate, bromide, nitrate, iodide, and thiocyanate), and organic anions (p‐aminobenzoic acid, p‐anilinesulfonic acid, sodium benzoate, pathalic acid, and salicylic acid) were also separated individually by anion‐exchange chromatography. Based on the multiple sites of the stationary phase, the column could separate 14 solutes containing the above series of analytes in one run. The dicationic imidazolium ionic liquid modified silica can interact with hydrophobic analytes by the hydrophobic C₆ chain; it can enhance selectivity to aromatic compounds by imidazolium groups; and it also provided anion‐exchange and electrostatic interactions with ionic solutes. Compared with a monocationic ionic liquid functionalized stationary phase, the new stationary phase represented enhanced selectivity owing to more interaction sites.     

Determination of N‐nitrosamines in cosmetic products by vortex‐assisted reversed‐phase dispersive liquid–liquid microextraction and liquid chromatography with mass spectrometry

A new analytical method for the simultaneous determination of trace levels of seven prohibited N‐nitrosamines (N‐nitrosodimethylamine, N‐nitrosoethylmethylamine, N‐nitrosopyrrolidine, N‐nitrosodiethylamine, N‐nitrosopiperidine, N‐nitrosomorpholine, and N‐nitrosodiethanolamine) in cosmetic products has been developed. The method is based on vortex‐assisted reversed‐phase dispersive liquid–liquid microextraction, which allows the extraction of highly polar compounds, followed by liquid chromatography with mass spectrometry. The variables involved in the extraction process were studied to obtain the highest enrichment factor. Under the selected conditions, 75 μL of water as extraction solvent was added to 5 mL of n‐hexane sample solution and assisted by vortex mixing during 30 s to form the cloudy solution. The method was successfully validated showing good linearity (0.5–50 ng/mL), enrichment factors up to 65 depending on the target compound, limits of detection values of 1.8–50 ng/g, and good repeatability (RSD < 9.8%). Finally, the proposed method was applied to different cosmetic samples. Quantitative relative recovery values (80–113%) were obtained, thus showing that matrix effects were negligible. The achieved analytical features of the proposed method, besides of its simplicity and affordability, make it useful to perform the quality control of cosmetic products to ensure the safety of consumers.     

Comprehensive analysis of pharmaceutical products using simultaneous mixed‐mode (ion‐exchange/reversed‐phase) and hydrophilic interaction liquid chromatography

Liquid chromatographic assays were developed using a mixed‐mode column coupled in sequence with a hydrophilic interaction liquid chromatography column to allow the simultaneous comprehensive analysis of inorganic/organic anions and cations, active pharmaceutical ingredients, and excipients (carbohydrates). The approach utilized dual sample injection and valve‐mediated column switching and was based upon a single high‐performance liquid chromatography gradient pump. The separation consisted of three distinct sequential separation mechanisms, namely, (i) ion‐exchange, (ii) mixed‐mode interactions under an applied dual gradient (reversed‐phase/ion‐exchange), and (iii) hydrophilic interaction chromatography. Upon first injection, the Scherzo SS C₁₈ column (Imtakt) provided resolution of inorganic anions and cations under isocratic conditions, followed by a dual organic/salt gradient to elute active pharmaceutical ingredients and their respective organic counterions and potential degradants. At the top of the mixed‐mode gradient (high acetonitrile content), the mobile phase flow was switched to a preconditioned hydrophilic interaction liquid chromatography column, and the standard/sample was reinjected for the separation of hydrophilic carbohydrates, some of which are commonly known excipients in drug formulations. The approach afforded reproducible separation and resolution of up to 23 chemically diverse solutes in a single run. The method was applied to investigate the composition of commercial cough syrups (Robitussin®), allowing resolution and determination of inorganic ions, active pharmaceutical ingredients, excipients, and numerous well‐resolved unknown peaks.     

Simultaneous determination of N‐ethylpentylone,dopamine, 5‐hydroxytryptamine and their metabolites in rat brain microdialysis by liquid chromatography tandem mass spectrometry

N‐Ethylpentylone (NEP) is a popular synthetic cathinone abused worldwide. To obtain more information about its pharmacokinetics and pharmacodynamics, a rapid, simple and sensitive liquid chromatography–tandem mass spectrometry method was developed for the determination of NEP, two important neurotransmitters, dopamine and serotonin, and their metabolites, including 3,4‐dihydroxyphenylacetic acid, 3‐methoxytyramine and 5‐hydroxyindole‐3‐acetic acid, in rat brain microdialysate. The analytes were separated on a Phnomenex Polar C₁₈ column, with a mobile phase of 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B) under gradient elution to shorten the total chromatographic run time. A triple quadruple mass spectrometer coupled with an electrospray ionization source in both positive and negative ion mode was used to detect the analytes. This method showed excellent accuracy (87.4–113.5%) and precision (relative standard deviation <15%) at three quality control levels. The limits of detection were 0.2 ng/mL for NEP and 0.2–50 nm for the others and good linearity was obtained. This study pioneered a method to integrate exogenous drugs and endogenous neurotransmitters as the drugs act on the same determination system, which means that this innovation can provide support for further study of the addictive effects of NEP or other synthetic cathinones on extracellular levels of dopamine and 5‐hydroxytryptamine.     

Some insights on the description of gradient elution in reversed‐phase liquid chromatography

The so‐called “fundamental equation for gradient elution” has been used for modeling the retention in gradient elution. In this approach, the instantaneous retention factor (k) is expressed as a function of the change in the modifier content (φ(t_s)), t_s being the time the solute has spent in the stationary phase. This approach can only be applied at constant flow rate and with gradients where the elution strength depends on the column length following a f(t?l/u) function, u being the linear mobile phase flow rate, and l the distance from the column inlet to the location where the solute is at time t measured from the beginning of the gradient. These limitations can be solved by using the here called “general equation for gradient elution”, where k is expressed as a function of φ(t,l). However, this approach is more complex. In this work, a method that facilitates the integration of the “general equation” is described, which allows an approximate analytical solution with the quadratic retention model, improving the predictions offered by the “linear solvent strength model.” It also offers direct information about the changes in the instantaneous modifier content and retention factor, and gives a meaning to the gradient retention factor.     

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography: Orthogonality in separation of Polygonum cuspidatum Sieb. et Zucc

Off‐line comprehensive two‐dimensional reversed‐phase countercurrent chromatography with high‐performance liquid chromatography was investigated in separation of crude ethanol extract from traditional Chinese medicinal herb Polygonum cuspidatum Sieb. et Zucc. Two‐dimensional contour plots for countercurrent chromatography with high‐performance liquid chromatography was obtained after comprehensive separation was completed. Total peak capacity was evaluated and approximately 810 peaks were obtained through a comprehensive two‐dimensional separation. A highly orthogonality of 52.23% and a large separation space occupancy of 88.86% were achieved. Meanwhile, it was found that several components could be well separated by countercurrent chromatography while they could not be separated by high‐performance liquid chromatography, and vice versa, which further indicated the orthogonality of the two separation methods. The off‐line comprehensive two‐dimensional countercurrent chromatography with high‐performance liquid chromatography provided a promising and powerful method for separation of complex natural products.     

Quantitative analysis of N‐acylphosphatidylethanolamine molecular species in rat brain using solid‐phase extraction combined with reversed‐phase chromatography and tandem mass spectrometry

A novel method for the sensitive and selective identification and quantification of N‐acylphosphatidylethanolamine molecular species was developed. Samples were prepared using a combination of liquid–liquid and solid‐phase extraction, and intact N‐acylphosphatidylethanolamine species were determined by reversed‐phase high‐performance liquid chromatography coupled to positive electrospray tandem mass spectrometry. As a result of their biological functions as precursors for N‐acylethanolamines and as signaling molecules, tissue concentrations of N‐acylphosphatidylethanolamines are very low, and their analysis is additionally hindered by the vast excess of other sample components. Our sample preparation methods are able to selectively separate the analytes of interest from any expected biological interferences. Finally, the highest selectivity is achieved by coupling chromatographic separation and two N‐acyl chain specific selected reaction monitoring scans per analyte, enabling identification of both the N‐acyl chain and the phosphatidylethanolamine moiety. The validated method is suitable for the reliable quantification of N‐acylphosphatidylethanolamine species from rat brain with a lower limit of quantification of 10 pmol/g and a linear range up to 2300 pmol/g. In total, 41 N‐acylphosphatidylethanolamine molecular species with six different N‐acyl chains, amounting to a total concentration of 3 nmol/g, were quantified.     

Porous silica particles grafted with an amphiphilic side‐chain polymer as a stationary phase in reversed‐phase high‐performance liquid chromatography

The amphiphilic polymer‐grafted silica was newly prepared as a stationary phase in high‐performance liquid chromatography. Poly(4‐vinylpyridine) with a trimethoxysilyl group at one end was grafted onto porous silica particles and the pyridyl side chains were quaternized with 1‐bromooctadecane. The obtained poly(octadecylpyridinium)‐grafted silica was characterized by elemental analysis, diffuse reflectance infrared Fourier transform spectroscopy and Brunauer–Emmett–Teller analysis. The degree of quaternization of the pyridyl groups on the obtained stationary phase was estimated to be 70%. The selective retention behaviors of polycyclic aromatic hydrocarbons including some positional isomers were investigated using poly(octadecylpyridinium)‐grafted silica as an amphiphilic polymer stationary phase in high‐performance liquid chromatography and results were compared with commercially available polymeric octadecylated silica and phenyl‐bonded silica columns. The results indicate that the selectivity toward polycyclic aromatic hydrocarbons exhibited by the amphiphilic polymer stationary phase is higher than the corresponding selectivity exhibited by a conventional phenyl‐bonded silica column. However, compared with the polymeric octadecylated silica phase, the new stationary phase presents similar retention behavior for polycyclic aromatic hydrocarbons but different retention behavior particularly for positional isomers of disubstituted benzenes as the aggregation structure of amphiphilic polymers on the surface of silica substrate has been altered during mobile phase variation.     

Quantitative determination of acidic hydrolysis products of Chemical Weapons Convention related chemicals from aqueous and soil samples using ion‐pair solid‐phase extraction and in situ butylation

Chemical warfare agents such as organophosphorus nerve agents, mustard agents, and psychotomimetic agent like 3‐quinuclidinylbenzilate degrade in the environment and form acidic degradation products, the analysis of which is difficult under normal analytical conditions. In the present work, a simultaneous extraction and derivatization method in which the analytes are butylated followed by gas chromatography and mass spectrometric identification of the analytes from aqueous and soil samples was carried out. The extraction was carried out using ion‐pair solid‐phase extraction with tetrabutylammonium hydroxide followed by gas chromatography with mass spectrometry in the electron ionization mode. Various parameters such as optimum concentration of the ion‐pair reagent, pH of the sample, extraction solvent, and type of ion‐pair reagent were optimized. The method was validated for various parameters such as linearity, accuracy, precision, and limit of detection and quantification. The method was observed to be linear from 1 to 1000 ng/mL range in selected ion monitoring mode. The extraction recoveries were in the range of 85–110% from the matrixes with the limit of quantification for alkyl phosphonic acids at 1 ng/mL, thiodiglycolic acid at 20 ng/mL, and benzilic acid at 50 ng/mL with intra‐ and interday precisions below 15%. The developed method was applied for the samples prepared in the scenario of challenging inspection.     

Determination of Cyanuric Acid in Milk Powder and Swimming Pool Water by Ion‐pair Reversed‐phase Liquid Chromatography

An ion‐pair reversed‐phase high‐performance liquid chromatographic method, using tetrabutylammonium bromide (TBAB) as ion‐pair reagent, has been developed for the analysis of cyanuric acid (CA) in milk powder and swimming pool water. It was found that the effect of the concentrations of ion‐pair reagent on the retention of cyanuric acid was different for standard solution and different real samples. The separation was carried out on a reversed‐phase C₁₈ column with 85:15 (V/V) water‐acetonitrile (ACN) containing different concentration of TBAB as mobile phase for different samples. The linear range of the calibration curve for CA was 0.1–100 mg·L^?1. The detection limits calculated at S/N=3 was 0.11 mg·L^?1 for the analysis of milk powder and 0.31 mg·L^?1 for the analysis of swimming pool water, respectively. The method was successfully applied to the analysis of CA in milk powder and swimming pool water.