Concentration Of Ion-pairing Agent Research Articles

Tetrabutylammonium HPLC analysis: shortcomings in the Ph. Eur. method.

Tetrabutylammonium is a phase-transfer agent commonly used in PET radiochemistry. Its toxicity makes its quantification mandatory. However, the official HPLC method for tetrabutylammonium analysis reported in the European Pharmacopoeia (Ph. Eur.) apparently fails to achieve the described separation in most new generation reverse-phase columns. The study highlights the differences in separation achievable by varying some of the chromatographic conditions, such as temperature, eluent composition and ion-pairing agent concentration. In the end, variations to the method within the limits allowed by the Ph. Eur. were not sufficient to overcome the problem, thus forcing to a more radical change of the organic component of the mobile phase.

The impact of ion-pairing reagents on the selectivity and sensitivity in the analysis of modified oligonucleotides in serum samples by liquid chromatography coupled with tandem mass spectrometry

Present study highlights the usage of various ion-pairing agents and their impact on the process of separation and ionization of oligonucleotides in the fortified human serum samples. What is more, retention studies involved different stationary phases, including: octadecyl, phenyl, pentafluorophenyl groups and ligands with embedded polar groups. It was proved that retention of oligonucleotides strongly depends on the alkyl chain branching in the structure of ion pairing reagent. Furthermore ion-pairing agents build of straight alkyl chain are more easily adsorbed on the stationary phase modified with octadecyl groups, while branching of alkyl chain caused more effective adsorption of studied compounds at phenyl groups compared to octadecyl ones. The lowest limit of quantification values were obtained for 5mMN,N-dimethylbutylamine, while the highest values are characteristic for hexylamine. Moreover it was shown that a 2-fold increase of ion-pairing agent concentration results in higher LOQ. The greatest sensitivity was obtained for 2.5mMN,N-dimethylbutylamine/150mM hexafluoroisopropanol. On the other hand Hypersil GOLD aQ column was the most appropriate in terms of time and separation effectiveness. The developed method was successfully used for the determination of studied oligonucleotides and their metabolites in human serum samples. The compounds were separated in just 3.5min with high sensitivity (0.09–0.16ng).

Effect of Mobile Phase pH on the Function of Other Optimization Parameters in an HPLC–ECD Assay of Biogenic Amines and Their Metabolites

We evaluated the effect of pH on other optimization factors for quantification of biogenic amines and metabolites by ion-pair reverse phase HPLC–ECD. Sequential changes were made in mobile phase pH, concentration of ion pairing agent and organic modifier to assess the primary function and secondary interaction of these parameters. The data show that pH adjustments within the range commonly reported alter the effects of other optimization factors on the retention of acid metabolites, but not neutral metabolites or the biogenic amines. The biogenic amine assay often requires fine adjustments to optimize retention of the target compounds. An understanding of the multiple effects of changes in mobile phase pH will facilitate optimization of this complex assay.

Ion-pair cloud-point extraction: a new method for the determination of water-soluble vitamins in plasma and urine.

A novel, simple, and effective ion-pair cloud-point extraction coupled with a gradient high-performance liquid chromatography method was developed for determination of thiamine (vitamin B1 ), niacinamide (vitamin B3 ), pyridoxine (vitamin B6 ), and riboflavin (vitamin B2 ) in plasma and urine samples. The extraction and separation of vitamins were achieved based on an ion-pair formation approach between these ionizable analytes and 1-heptanesulfonic acid sodium salt as an ion-pairing agent. Influential variables on the ion-pair cloud-point extraction efficiency, such as the ion-pairing agent concentration, ionic strength, pH, volume of Triton X-100, extraction temperature, and incubation time have been fully evaluated and optimized. Water-soluble vitamins were successfully extracted by 1-heptanesulfonic acid sodium salt (0.2% w/v) as ion-pairing agent with Triton X-100 (4% w/v) as surfactant phase at 50°C for 10 min. The calibration curves showed good linearity (r(2) > 0.9916) and precision in the concentration ranges of 1-50 μg/mL for thiamine and niacinamide, 5-100 μg/mL for pyridoxine, and 0.5-20 μg/mL for riboflavin. The recoveries were in the range of 78.0-88.0% with relative standard deviations ranging from 6.2 to 8.2%.

Robustness Testing in HPLC Analysis of Clarithromycin, Norfloxacin, Doxycycline, Tinidazole and Omeprazole in Pharmaceutical Dosage forms Using Experimental Design

Robustness tests were performed on a gradient RP-HPLC method that had been developed for rapid simultaneous separation and determination of clarithromycin, norfloxacin, doxycycline, tinidazole and omeprazole in their pharmaceutical dosage forms. Separation was carried out on a 250 x 4.6 mm (i.d.), 5µm ODS column (Inertsil, Tokyo, Japan) and multiwavelength overlay chromatograms at max of 210 and 310 nm were used for quantitative analysis. The standard curve was linear in the concentration range of 100 – 900, 5 – 150, 20 – 220, 10 - 150 and 0.5 - 110 µg ml-1 for clarithromycin, norfloxacin, doxycycline, tinidazole and omeprazole, respectively. Robustness tests were performed using twolevel fractional factorial designs with resolution III, so only main effects can be estimated. Factors chosen for this study were the column temperature (˚C), pH, ion pairing agent concentration (g/l), phosphate buffer concentration (g/l), the fraction of mobile phase B (B% start) at 4 min of the gradient and fraction of mobile B (B% end) at 7 min of the gradient run. The significance of the factor effects was determined statistically, using algorithm of Dong in the calculation of critical effects, and graphically, by means of half-normal plots. Non-significant intervals for significant factors and system suitability limits were calculated based on robustness tests' results. The method was validated according to ICH guidelines. The proposed method was found to be accurate, reproducible, and consistent which is useful for the routine determination of clarithromycin, norfloxacin, doxycycline, tinidazole and omeprazole in combined mixtures and pharmaceutical dosage forms.

Preconcentration and Determination of Cadmium in Water and Food Samples byin situSurfactant-Based Solid-Phase Extraction and Flame Atomic Absorption Spectrometry

In situ surfactant-based solid-phase extraction (ISS-SPE) is proposed as a preconcentration procedure for the determination of cadmium in water and food samples by flame atomic absorption spectrometry. In the present work, cetyltrimethylammonium bromide was used as a cationic surfactant, hexafluorophosphate ion as an ion-pairing agent and 4-benzylpiperidinedithiocarbamate potassium salt (K-4-BPDC) as a chelating agent. Several variables that affect the extraction efficiencies such as pH, type and amount of surfactant, concentration of ion-pairing agent, concentration of chelating agent, ionic strength and extraction time were investigated and optimized. After optimization of the complexation and extraction conditions, an enrichment factor of 40 was obtained. The calibration curve was linear in the range of 1-50 µg L-1 and the limit of detection was 0.3 µg L-1. The relative standard deviation for 20 µg L-1 of cadmium was lower than 2.2%. Validation of the methodology was performed by standard addition method and analysis of certified reference materials. The method was successfully applied to the determination of cadmium in various food and water samples.

Analysis of Grape Proteins from Wines by Perfusion Reversed-Phase High-Performance Liquid Chromatography

Concentration and characteristics of grape proteins can strongly influence the quality of the wine, therefore the availability of reliable information of these biomolecules are crucial. This paper describes, for the first time, a simple and fast reversed-phase high-performance liquid chromatography (RP-HPLC) method enabling the separation and quantification of grape proteins from wines. Several factors, such as gradient elution, column temperature, detection wavelength, type and concentration of ion-pairing agent and the conditions for the sample preparation were optimised. The proposed method enables a separation of the grape proteins from wines in only 7 min using a linear binary gradient of water/acetonitrile/0.1 % trifluoracetic acid in four steps at a flow rate of 3 mL/min with a column temperature of 65 °C and UV detection at 210 nm. The method was validated and applied to the quantification of grape proteins in white and rose wines. The potential of the method was also demonstrated by its application to the differentiation of wine samples with different aging over lees and grape varieties.

Combination of In Situ Surfactant-based Solid Phase Extraction and Central Composite Design for Preconcentration and Determination of Manganese in Food and Water Samples

A new kind of solid phase extraction (SPE), which we named in situ surfactant-based solid phase extraction (ISS-SPE), represents a simple, selective and rapid method for preconcentration and determination of manganese from food and water samples. This method has distinct advantages: extraction times are short and recoveries are high; further, we can see formation of fine particles of large specific surface and their good dispersion in the solution. In this work, a small amount of cationic surfactant, n-dodecyltrimethylammonium bromide (DTAB) was injected into the water sample containing Mn ions, which were complexed by 1-(2-pyridylazo)-2-naphthol (PAN). After shaking, a little volume of NaPF6 as an ion-pairing agent was added into the solution by a microsyringe. After preconcentration, the settled phase was dissolved in a specific volume of ethanol and then aspirated into the flame atomic absorption spectrometer by using a homemade microsample introduction system. The effective parameters such as pH, concentration of surfactant, concentration of chelating agent, concentration of ion-pairing agent and effect of salt concentration were optimized by a fractional factorial design to identify the most important parameters and their interactions, and central composite methodology was used to achieve the optimum point of effective parameters to the response. Under the optimum conditions, preconcentration of 10 ml of the sample solution permitted detection of 0.88 μg l−1 with enhancement factor of 45.6, and the relative standard deviation (RSD) for five determinations of Mn ions was 3.5%. The developed method was applied to the determination of trace manganese in various real samples with satisfactory results.

A STUDY ON INTERACTION OF ION-PAIRING AGENT IN SEPARATION OF HUMAN INSULIN AND GLYCOSYLATED HUMAN INSULIN USING REVERSE PHASE LIQUID CHROMATOGRAPHY

Glycosylated proteins formed during expression are present as impurities in yeast based recombinant production of Human Insulin (HI). These closely related glycosylated impurities pose challenge in purification of HI by RP-HPLC. In this work, separation of glycosylated Human Insulin (gHI) and HI in presence of other HI related impurities under preparative loading conditions was studied at pH between 2.8 to 4.0. Sodium salts of acetate, citrate, formate, perchlorate, and succinate were used as ion-pairing agents in mobile phase. The study was performed by varying the concentration of ion-pairing agent, pH of mobile phase, and HI loading on the column. The effect of ion-pairing agent, in conjunction with pH, on resolution of gHI and HI was evaluated based on recovery, purity of HI, and percent reduction of gHI. Ion-pair formation of sodium perchlorate in the presence of acetonitrile as the organic modifier resulted in effective separation of gHI and HI yielding HI purity of 98.5% under preparative loading conditions (5 to 15 g/L). In order to comply with the purity and impurity specifications as per pharmacopeia, development of additional purification methods would be required, based on analysis results of the product using pharmacopeia methods. Supplemental materials are available for this article. Go to the publisher's online edition of Journal of Liquid Chromatography & Related Technologies to view the free supplemental file.

Pyridinium aldoxime analysis by HPLC: the method for studies on pharmacokinetics and stability

Reversed-phase separation of various pyridinium aldoximes requires a certain concentration of ion-pairing agent, as their chemical structures contain two quaternary amines in the pyridinium ring. Adequate mobile phase is scouted on the basis of retention of pyridinium aldoxime (using the graph of k' versus concentration of an ion-pairing agent) compared to the chromatogram of the background peaks originated from the homogenate. Change in the ion-pairing agent concentration was more expressed for the elution of K-203 than that of the background peaks from the serum, brain and cerebrospinal fluid. Stability of K-203 was investigated using HPLC. Determination of K-203 in tissue samples requires homogenization using either trichloroacetic acid or perchloric acid. Fast degradation takes place at acidic pH. Adjusting pH to neutral in the possible shortest time frame helps to avoid degradation. Degradation of K-203 was easily followed by HPLC separation and monitoring the elution with an ultraviolet absorbance detector at 276 nm. Amperometric detection indicates only the decrease of K-203 content.

RP-HPLC ANALYSIS OF HYDROPHOBIC ALENDRONATE AMIDATED DERIVATIVES

The reversed phase high performance liquid chromatography (RP-HPLC) was evaluated for its application in the analysis of the quantiation of the alendronate derivatives. The anti-phase Agilent CLC-ODS C18 column was used at the temperature of 50°C. The RP-HPLC trace was monitored with a differential refractive index detector for the lack of ultraviolet (UV) absorbing groups in the derivative molecule. The mobile phase was an aqueous solution mixture of MeOH and H2O at pH 7.2, containing small amounts of dodecoulde tributyl bromide and disodium ethylente diamine tetraacetic acid. The chromatographic conditions, such as the concentration of ion-pairing agent, pH, and methanol content in mobile phase, were studied. This RP-HPLC method provides high separation and specificity to derivative analysis. In quantitative analysis, the method showed satisfactory precision (less than 0.62%) and recovery (higher than 98.3%), good linearity (r2 = 0.9994) with the linear range of 0.5 ∼ 250 μg·mL−1. The limit of quantification (0.5 μg·mL−1) and limit of detection (0.2 μg·mL−1) were also achieved.

Quantitative profiling of nucleotides and related phosphate-containing metabolites in cultured mammalian cells by liquid chromatography tandem electrospray mass spectrometry

A method has been developed for the quantitative profiling of over twenty nucleotides and related phosphorylated species using ion-pair reversed-phase liquid chromatography hyphenated to negative ion tandem electrospray mass spectrometry. The influence of mobile phase pH and ion-pairing agent concentration were assessed to optimise separation and peak shapes. Full quantitative analysis was obtained for the nucleotides by reference to structurally related calibration standards. The developed method was applied to profile changes in nucleotides and related compounds in monolayer cultured Chinese hamster ovary (CHO) cells expressing the β 2 adrenoceptor when exposed to pharmacological stimuli. These experiments demonstrate the potential of the LC–MS/MS method to detect changes in nucleotide drug targets as well as the simultaneous monitoring of levels of other nucleotides.

A reversed-phase ion-interaction chromatographic method for in-vitro estimation of the percutaneous absorption of water-soluble UV filters

An analytical method based on ion-interaction chromatography with UV detection for simultaneous in-vitro estimation of the percutaneous absorption of the most used water-soluble UV filters in sunscreen cosmetics is proposed. These UV filters were phenylbenzimidazole sulfonic acid, disodium phenyl dibenzimidazole tetrasulfonate, benzophenone-4, and terephthalylidene dicamphor sulfonic acid. The methodology is based on applying the sunscreen containing the target UV filters to human epidermis in a diffusion cell. Analytes are determined in the receptor solution. To ensure skin integrity, screening of the cells was carried out by analytical determination of a marker. Analytical variables such as percentage ethanol, concentration of ion-pairing agent, pH of the mobile phase, and temperature were studied in order to achieve high resolution of the chromatographic peaks in the lowest possible time of analysis. The conditions selected consisted of a mobile phase composed of 35:65 (v/v) ethanol-ammonium acetate buffer solution (pH 4, containing 50 mmol L(-1) tetra-n-butylammonium bromide). The chromatographic determination was carried out with the analytical column at 50 degrees C. UV detection was carried out at the maximum absorption wavelength for each analyte. The limit of detection (3s(y/x)/b) ranged from 16 to 65 ng mL(-1), depending on the analyte.

Monolithic Supports for the Characterization of Commercial Maize Products Based on Their Chromatographic Profile. Application of Experimental Design and Classification Techniques

Modern analytical techniques based on the use of RP-HPLC with monolithic stationary phases and the application of experimental design and classification tools have been applied to the analysis of maize proteins. Solubilization conditions of maize proteins and separation conditions (temperature, detection wavelength, type and concentration of ion-pairing agent, and gradient) were optimized. The elution gradient was optimized by the application of experimental design techniques. The optimized method consisted of a linear binary gradient of water/acetonitrile/0.1% trifluoroacetic acid in three steps at a flow rate of 3 mL/min with a column temperature of 35 degrees C and UV detection at 280 nm. The developed method enabled the separation of maize proteins in an analysis time close to 8 min. Moreover, this is the first time that commercial maize products have been characterized by the use of multivariate classification techniques.

Development of a perfusion reversed-phase high performance liquid chromatography method for the characterisation of maize products using multivariate analysis

A perfusion reversed-phase high performance liquid chromatography (RP-HPLC) method has been designed to allow rapid (3.4 min) separations of maize proteins with high resolution. Several factors, such as extraction conditions, temperature, detection wavelength and type and concentration of ion-pairing agent were optimised. A fine optimisation of the gradient elution was also performed by applying experimental design. Commercial maize products for human consumption (flours, precocked flours, fried snacks and extruded snacks) were characterised for the first time by perfusion RP-HPLC and their chromatographic profiles allowed a differentiation among products relating the different technological process used for their preparation. Furthermore, applying discriminant analysis makes it possible to group the samples according with the technological process suffered by maize products, obtaining a good prediction in 92% of the samples.

Reversed-phase liquid chromatography of metal chelates of 4-(2-pyridylazo) resorcinol: retention model and its verification

Based on the concept of limiting capacity factors, a semi-quantitative retention model was proposed to describe the chromatographic behaviour of metal chelates with 4-(2-pyridylazo) resorcinol in reversed-phase and ion-pair reversed-phase systems. Separations were carried out on octadecyl-bonded stationary phase with mobile phase consisted of an aqueous buffer — methanol mixture; tetrabutylammonium hydroxide was used as ion-pairing agent in ion-pair reversed-phase chromatography. Experimental dependencies of the capacity factors on the mobile phase characteristics (pH, methanol concentration, concentration of ion-pairing agent) were measured for nickel, iron, copper and cobalt chelates, and confronted with the retention model. A good agreement was found between experimental data and the dependencies predicted from the retention model for the chelates of Ni 2+, Fe 2+ and Cu 2+, whereas the cobalt chelate exhibited anomalous behaviour, which can not be fully explained with the aid of the proposed model. The chelate retention is strongly affected by the presence of methanol in mobile phase in a similar way as was commonly observed in the reversed-phase chromatography of organic substances. The buffer concentration (ionic strength of mobile phase), on the other hand, has almost negligible effect on the chelate retention. Side equilibria, such as acid–base or ion-pairing, can be used to manipulate effectively the chelate retention and separation; these equilibria are involved in the retention model.

Ion-pair liquid chromatography–electrospray mass spectrometry for the analysis of cyclic nucleotides

An LC–MS method has been developed combining ion-pair chromatography with an electrospray interface linking microbore and capillary HPLC to mass spectrometry. Separation of cyclic nucleotides on C 18 reversed-phase columns, using tetrabutylammonium bromide as an ion pairing agent was evaluated with different mobile phase compositions. It was found that low ion-pairing agent concentration (50–500 μ M) used in combination with low flow-rates (5–10 μl min −1) allowed the system to operate for up to several days without observing a reduced signal caused by source pollution. The loss of sensitivity expected in ion-pair chromatography could be remedied by using a 2-propanol coaxial sheath flow. Optimal conditions for negative ion electrospray resulted in a linear detection response in the femtomole to picomole range. Using biological samples this method was evaluated and compared with a classical ion-suppression RP-HPLC method using UV detection.

Simultaneous measurement of serotonin and paroxetine in rat brain microdialysate by a single-pump column-switching technique.

Simultaneous quantitation of paroxetine and serotonin in rat brain microdialysate is presented as a means to study the neuropharmacokinetics and neuropharmacodynamics of paroxetine, a selective serotonin reuptake inhibitor. In order to achieve this objective, a single-pump column-switching technique was developed. Optimization of the mobile phase in terms of the concentration of ion-pairing agent, pH of mobile phase, temperature of the stationary phase and concentration of organic modifier was investigated and a single mobile phase was developed for both separations. The design was such that the switching valve employed column I (50 mm length) and column II (250 mm length) in series in position A. At 15.3 min, the valve was switched to position B, in which the flow of the mobile phase was directed only through the short column (column I). A flow gradient program was used to increase the flow-rate from 0.125 ml/min to 0.4 ml/min, which enabled a reduction in total analysis time to less than 20 min. The limits of detection for serotonin and paroxetine were 6 fmol and 300 fmol, respectively. The accuracy of the method demonstrated percent differences from spiked samples that were within 12.5% and the precision was found to be within 10% R.S.D.

High-performance size-exclusion chromatography of polypeptides on a TSK G2000SW column in acidic mobile phases

Polypeptides subjected to high-performance liquid chromatography on a TSK G2000SW column in acidic mobile phases of low ionic strength underwent a combination of size exclusion and ion exclusion from the matrix. The distribution coefficient ( K d) of each polypeptide was dependent upon the concentration of ion-pairing agent present and increased as the concentration of ion-pairing agent increased. The fractionation range of the column could thus be manipulated by altering the concentration of acid in the mobile phase. The nature of the ion-pairing acid, in terms of hydrophilic or hydrophobic ion pairing, also had an effect on the chromatography, such that hydrophobic ion-pairing agents lead to increased K d values, especially in the case of the most basic polypeptides. To account for these results it is proposed that the TSK GSW matrix is cationic at low pH, and that ion pairing suppresses the ion exclusion experienced by cationic polypeptides under these conditions.

The Separation of Bilirubin, Photobilirubin and their Major Isomers by Ion-Pair High Pressure Liquid Chromatography

Abstract The separation of the three major isomers and the main photoproducts of bilirubin by Ion-Pair High Pressure Liquid Chromatography is described. Chromatographic procedures found in the literature proved difficult to reproduce, had unacceptably long elution times (over 2 hours), required complex gradients or two or more columns. As these reports provided no data to show why specific chromatographic conditions were chosen an optimization study was carried out. The separation is shown to be dependent on pH, organic content and buffer concentration of the mobile phase, ion-pairing agent concentration and injection solvent. A separation, carried out in less than thirty minutes on octadecyl silica using isocratic conditions with detection at 455 nm is demonstrated. This technique will be used in the elucidation of the mechanism involved in phototherapy treatment of neonatal jaundice.