Ion-pair Reversed-phase Liquid Chromatography Research Articles

Chemiluminescence detection coupled to liquid chromatography for the determination of penicillamine in human urine

An on-line liquid chromatography (LC) chemiluminescence detection system is proposed for the determination of penicillamine in human urine. Ion-pair reversed-phase LC using octane sulphonic acid as ion-pairing reagent allows the separation of penicillamine from the urine matrix. The Chemiluminescence reaction of penicillamine with Ce(IV) in sulphuric acid sensitized by quinine is suggested for sensitive penicillamine detection in a post-column mixing set-up. The Chemiluminescence response was linearly related to the concentration of penicillamine in the range 2.0–2000 μmol 1 −1 ( r = 0.9994, peak height, and 0.9997 peak area, n = 10) with a detection limit ( S N = 3) of 1.0 μ mol 1 −1 (100 pmol per injection). Relative standard deviations were less than 5% ( n = 10) for 10–50 μ mol 1 −1. The excretion rates of penicillamine in human urine of three volunteers after oral penicillamine administration were determined in the present study.

Glutathione and N-acetylcysteine reduce gastric mucosal blood flow in rats.

Glutathione has been studied as a possible mediator in gastric mucosal protection and healing, but its extracellular function is not fully understood. This study evaluates blood flow changes in normal gastric mucosa secondary to glutathione modulation under stable central hemodynamic conditions. Thiol substances were quantified by reverse-phase ion-pair liquid chromatography and fluorescence detection. Central hemodynamics remained stable when glutathione and N-acetylcysteine were administered in a dose of 0.5 mmol/kg. Higher doses than 0.5 mmol/kg of glutathione and N-acetylcysteine caused unstable hemodynamics. Glutathione (0.5 mmol/kg intravenously) and N-acetylcysteine (0.5 mmol/kg intravenously) reduced corpus mucosal blood flow by 28% and 26% (P < 0.0005), respectively, and glutathione reduced antral mucosa blood flow by 22% (P < 0.01). L-Buthionine-[S,R]-sulfoximine (2 mmol/kg intravenously) did not effect gastric mucosal blood flow. Cysteine content in mucosa and plasma increased while mucosal glutathione levels were largely unchanged after administration of reduced glutathione and N-acetylcysteine. Plasma glutathione only increased after injection of glutathione. L-Buthionine-[S,R]-sulfoximine reduced the glutathione level in both plasma and mucosa. We conclude that glutathione and N-acetylcysteine reduce gastric mucosal blood flow and that the effect may be related to increased cysteine levels in plasma or mucosa.

Microbore liquid chromatography analysis of monoamine transmitters.

Ion-pair reversed-phase liquid chromatography (LC), as introduced by Johansson et al. in 1978 (1) is still the most widely used separation technique for analysis of the biogenic amines. It is usually coupled with electrochemical detection (ECD). The use of microbore LC was introduced by Scott and Kucera in 1976 (2). These systems use columns with an internal diameter (id) of 1 mm or less. Microbore LC provides a significant advantage over conventional LC: The sample injected on to a microbore column is diluted by about 20-fold less than when injected on to a “normal” bore (e.g., 4.6 mm id) column. In this way, the compounds of interest become easier to detect. Microbore LC offers several other advantages:

Equilibrium and Kinetic Studies on Ligand Exchange Reaction of Aluminum(III)-5-sulfoquinoline-8-ol Complex with Fluoride Ion by Reversed-Phase Ion-Pair Liquid Chromatography.

The reversed-phase ion-pair liquid chromatographic behavior of the complex of aluminum ion with 5-sulfoquinoline-8-ol (HQS) was investigated on the column packed with a polystyrene-divinylbenzene copolymer with the mobile phase containing fluoride ion. It was found that the fluoride ion replaces one or two HQS molecules from the aluminum-HQS complex to form the mixed ligand complexes, which resulted in the appearance of two or more peaks in the chromatograms obtained for aluminum ion. The compositions of the mixed ligand complexes were estimated from the dependence of the areas of the peaks on the mobile phase concentrations of HQS and fluoride ion. The equilibrium and rate constants for the ligand exchange reaction were also evaluated and the reaction mechanism was presented. Aluminum ion could be completely separated from gallium ion by utilizing the ligand exchange reaction.

Redox Reaction Catalyzed by a Porous Graphite Carbon Packing and its Application to Selectivity Enhancement of High-performance Liquid Chromatography Separation of Metal Complexes

A new HPLC method was developed for the selective separation of metal complexes by using a redox reaction as a secondary chemical equilibrium. In a study of the reversed-phase ion-pair liquid chromatography of trans-1,2-diaminocyclohexanetetraacetate (DCTA) complexes of transition metal ions, it was noted that the redox reaction between the divalent and trivalent cobalt complexes were extremely accelerated by using a porous graphite carbon (PGC) column. This finding was ascribed to the catalytic effect of the PGC. The retention of Co–DCTA complex can be modified by treating the PGC column with the eluent containing sodium sulfite. The cobalt complex was completely separated from 1000-fold molar excess of the BiIII, FeIII, CuII, and NiII complexes.

Equilibrium and Kinetic Studies of Ligand-Exchange Reaction of Aluminum(III)-5-Sulfoquinoline-8-ol Complex with Phosphate Ion by Reversed-Phase Ion-Pair Liquid Chromatography

The reversed-phase ion-pair liquid chromatographic behavior of complexes of alummum(lll) with 5-sulfoquinoline-8-ol (HQS) on a column packed with a polystyrenedivinylbenzene copolymer is investigated with eluents that contain phosphate buffer. The aluminumdll)-HQS complex results in two peaks on the chromatograms; the heights of the peaks strongly depend on the pH of the eluent, the concentrations of HQS and phosphate buffer in the eluent, and the column temperature. The results obtained are interpreted according to the ligand-exchange reaction of aluminum(lll)-HQS with H 2 PO 4 - or HPO 4 2 in the eluent. The compositions of the mixed ligand complexes are determined, and the equilibrium and rate constants for the ligand-exchange reaction are estimated.

Determination of Lincomycin Residue in Salmon Tissues by Ion-Pair Reversed-Phase Liquid Chromatography with Electrochemical Detection

A method is described for detecting and quantitating lincomycin residue in salmon muscle and skin tissues by ion-pair reversed-phase liquid chromatography (LC) with electrochemical detection at +0.9 V. Lincomycin was extracted from tissues by homogenizing with 0.01 M KH2PO4 buffer (pH 4.5) and centrifuging the mixture. Water-soluble proteins were precipitated by adding sodium tungstate and sulfuric acid and removed by centrifugation. The buffer extract was then passed through a C18 solid-phase extraction cartridge. Lincomycin was eluted with 50% acetonitrile in water, and the eluate containing lincomycin was extracted with ethyl acetate. After the solvent had evaporated, the residue was redissolved in mobile phase and analyzed by LC. The method had a limit of detection of 7 ng/g lincomycin for salmon muscle and 12 ng/g for salmon skin. The limit of quantitation was 17 ng/g for salmon muscle and 24 ng/g for salmon skin. Average recoveries of lincomycin spiked at 50, 100, and 200 ng/g were > or = 85% for salmon muscle and > or = 80% for salmon skin.

Ion-pair reversed-phase high-performance liquid chromatography of algal chlorophylls

The Chromatographic behaviour of algal chlorophylls under ion-pair reversed-phase liquid chromatography conditions has been tested either on monomeric and polymeric octadecylsilica columns. Three five-carbon amino compounds (pentylamine, piperidine and pyridine), differing in their molecular shapes and chemical properties, have been used as counterions. The monomeric octadecylsilica column did not allow the separation of chlorophylls c 1 and c 2 with any amine, but separated chlorophyllide a, chlorophyll c 3 and Mg-divinylpheoporphyrin a 5 when pyridinium acetate was used as ion pair agent. The polymeric octadecylsilica column separated chlorophylls c 1 and c 2 with the three counterions, achieving the highest retention and overall separation of acidic chlorophylls when pyridinium was employed as counterion.

Arsenic Speciation by Ion-Pair Reversed-Phase Liquid Chromatography with Coupled Amperometric and Ultraviolet Detection

An ίοη-pair reversed-phase liquid chromatographic method is used to separate As(lll), As(V), arsenobetam, arsenocholin, monomethylarsonic, and dimethylarsonic acids in a single run on a common C 18 stationary phase with coupled amperometric and ultraviolet detection. As(III) is not separated from arsenobetam, but only As(III) is detected by amperometry. The calibration curves of the tested species are linear from 0.5 to 3 μmol/L. Coefficient of variation values for intra-assay precision range between 2.5% and 9.2%. The limit of quantitation ranges from 0.1-1 μmol/L, depending on the species concerned. was set at 200 nm. The same authors tried to separate six different species (Asbet, As[III], DMA, MMA, Aschol and As[V]) by ion pairing with tetrabutylammonium hydroxyde (TBAH) (16), but the separation of As(III) from Asbet could not be achieved. The au­ thors proposed using a stationary phase and an alkaline mobile phase (pH 9.0), thus allowing dissociation of arsenious acid (pKa 9.3) and its separation from arsenobetam (17). Nevertheless the resolution of these two species was not satisfactory. Due to the rel­ ative failure of these studies, we decided to try differentiation of the various arsenical species by combining ultraviolet and am­ perometric detection after ion-pair chromatographic separation.

Studies of the Stability of a Substituted Phosphorothioate Using an Improved High Performance Liquid Chromatography Assay Coupled with Electrochemical Detection

Abstract A sensitive, reproducible and rapid HPLC assay was developed for preformulation and stability studies of a potential radioprotector, S-2-(3-methylaminopropylamino) ethyl-phosphorothioate, WR-3689. This method is based on the elution of WR3689 by high performance ion-pair reverse phase liquid chromatography using an electrochemical detector with an oxidation potential set at + 0.19 v. The detector response was found to be linear over the concentration range of 0.02–20 μg/mL with an absolute limit of quantitation of 170 pg. The coefficient of variation ranged from 0.75–6.52% for the interday reproducibility. This HPLC assay was employed to determine the stability of WR-3689 at various pH (2, 4, 7, 10 and 12) and temperatures (22 °C, 35 °C, 43 °C and 100 °C). Results of the stability studies indicated that WR-3689 is more stable in basic than in acidic aqueous solutions. For example, the half-life of WR-3689 at 45 °C was 13.8 days at pH 12 as compared to 12.7 h at pH 4. With regard to the effect of ...

Determination of FCE 26644, a new polysulphonated derivative of distamycin A, in monkey plasma by reversed-phase ion-pair high-performance liquid chromatography with ultraviolet detection

A sensitive and selective ion-pair high-performance liquid chromatographic method for the determination of 7,7′-{carbonylbis[imino- N-methyl-4,2-pyrrolecarbonylimino- N-methyl-4,2-pyrrole)carbonylimino]- bis (1,3-naphthalenedisulphonic acid), tetrasodium salt in monkey plasma has been developed. The compound and internal standard (bromphenol blue) were extracted from plasma samples with methylene chloride (twice) after deproteination with acetonitrile and addition of the ion-pairing agent (tetrabutylammonium hydroxide). The combined organic phases were dried, the residue dissolved in the mobile phase and then analysed by reversed-phase ion-pair liquid chromatography under isocratic conditions. The HPLC analysis time was about 20 min. Quantitation was achieved by UV detection at 323 nm. The linearity, precision and accuracy of the method were evaluated. The limit of quantitation was 0.3 μg/ml plasma. No interference from blank monkey, mouse, rat, dog and human plasma was observed. The suitability of the method for in vivo samples was ckecked by analysis of plasma samples drawn from three male cynomolgus monkeys that had received a 20 mg/kg single i.v. dose of the test compound.

Half-fraction and full factorial designs versus central composite design for retention modelling in reversed-phase ion-pair liquid chromatography

In a previous paper (J. O. De Beer, C. V. Vandenbroucke and D. L. Massart, J. Pharm. Biomed. Anal., 12, (1994) 1379–1396) liquid chromatographic (LC) retention modelling of the cough-syrup compounds methyl para-hydroxybenzoate (MPHB) and propyl para-hydroxybenzoate (PPHB), phenylephrine hydrochloride (PE) and chlorphenamine maleate (CPM) was studied using a face-centred central composite design. It is examined whether smaller half-fractional and full factorial designs with fewer experiments tend to reliably predict retention times of the latter compounds as well. Simplified regression modelling, however, neglecting more first-order and interactive effects and disregarding pure second-order effects, has to be set up. These smaller designs finally satisfy the prediction of the retention of MPHB, PPHB and PE also. Retention prediction of CPM is much less accurate. CPM has a p K a value of 4.0, which is encompassed by the examined mobile phase pH limits 3.0 and 5.0. Since the largest retention shifts occur near the p K a value, retention prediction in this area becomes more complex. CPM retention modelling from a full factorial design is useful if the mobile phase pH is fixed at 5.0 for methanol as well as for acetonitrile as organic modifers. The full factorial design, applied with acetonitrile as organic modifer, enables the selection of suitable LC parameter combinations for fast and complete separation of the four compounds in cough-syrup analysis.

Determination of Dissolved and Adsorbed EDTA Species in Water and Sediments by HPLC

This paper describes a method for determining EDTA species in various environmental samples at low molar concentrations by high-performance liquid chromatography (HPLC). Distinction between Fe(III)EDTA and all the other species can be made. NiEDTA can be detected semiquantitatively. The fraction of EDTA adsorbed to suspended particles or to sediments can be determined after desorption with phosphate. After complexation with Fe(III), the EDTA is detected by reversed-phase ion-pair liquid chromatography as the Fe(III)EDTA complex at a wavelength of 258 nm. The behavior of a variety of metal-EDTA complexes during analysis was checked. Determination of different EDTA species (Fe(III)EDTA, NiEDTA, and adsorbed EDTA) is possible in river water, groundwater, and effluents from wastewater treatment plants. Fe(III)EDTA was found to be the main species, at 30-70%; NiEDTA was <10% in most of the samples. Adsorbed EDTA was detected in suspended particles from rivers and wastewater treatment plants and in sediment cores from a lake. The method is suitable for a variety of different samples with different concentration ranges.

Simultaneous analysis of tiaramide metabolites in horse urine and plasma by solid-phase extraction and reversed-phase ion-pair liquid chromatography.

A simple method for the simultaneous analysis of tiaramide (TRA) metabolites in the horse is described. The sample preparation method using a Bond-Elut PH cartridge and stepwise elution with ice-cold, 30% aqueous methanol followed by additional methanol is effective for recovering the metabolites with different properties. The extraction method gives good recoveries (greater than 80%) and reproducibility. Each metabolite is well separated by high-performance liquid chromatography using an octadecyl-type column of polymer-based packing with a solvent system of 20 mM phosphate buffer (pH 6.5)-acetonitrile-tetrahydrofuran (6:1:0.2) containing a quaternary ammonium salt as an ion-pairing reagent. The detection limit of each metabolite is in the range of 20-80 ng/ml. The major metabolites of TRA in equine urine and plasma are N-acetic acid (TRAA) and N-oxide. The N-oxide of TRAA is detected as a metabolite in the urine. A dealkylation product PER) is detected as a minor metabolite. Unchanged TRA is detected only at low concentrations in the urine. Several minor metabolites of DER or TRAA or both are detected in the urine.

On-line preconcentration of selenium(IV) and selenium(VI) in aqueous matrices followed by liquid chromatography-inductively coupled plasma mass spectrometry determination

A study was carried out for the determination of selenite and selenate from water matrices using ion-pairing reversed-phase or anion exchange liquid chromatography followed by determination with inductively coupled plasma mass spectrometry (LC-ICP-MS), which used conventional pneumatic nebulization as sample introduction technique. The detection limits and background levels for the anion exchange chromatography were lower than those for the ion-pairing separation method. Based on the optimum conditions obtained with a 25 μl sample injection loop, a preconcentration column was coupled on-line with the anion exchange LC-ICP-MS system. The capability of this simple on-line preconcentration procedure was demonstrated for the analysis of various water matrices spiked with selenite and selenate including high salt-containing water, river, tap, and well water samples. The detection limits (3σ) obtained by preconcentration of 2–10 ml of the high salt-containing water and other aqueous matrices were 0.16–0.42 and 0.08–0.19 μg l −1 of Se for selenite and selenate, respectively. These relative detection limits were ca. two orders of magnitude better than those obtained with a 25 μl sample loop.

Determination of Phenol Sulfone, Phenol Sulfoxide, and Phenol Sulfonic Acid Metabolites of Fenamiphos in Soil by Liquid Chromatography

Abstract An analytical method has been developed to quantitate fenamiphos phenol sulfone, phenol sulfoxide, and phenol sulfonic acid in soil. Both control and analyte-fortified soil samples from Fresno, CA, were extracted with methanol–water (2 + 1) and partitioned with ethyl acetate. Fenamiphos phenol sulfone and phenol sulfoxide were determined by re-versed-phase liquid chromatography (LC) with UV detection at 240 nm after cleanup by silica solid-phase extraction. Fenamiphos phenol sulfonic acid was determined by reversed-phase ion-pairing LC with UV detection at 240 nm after cleanup by amino solid-phase extraction. Recoveries of fenamiphos phenol sulfoxide ranged from 88.2 to 111.0%, with an average of 97.4%. Recoveries of fenamiphos phenol sulfone ranged from 101.6 to 107.0%, with an average of 104.6%. Recoveries of fenamiphos phenol sulfonic acid ranged from 76.0 to 99.9%, with an average of 86.0%. Responses for analysis of analytes in both solvent and soil matrix were linear over the tested range of 10 to 500 ppb. Limits of determination of each analyte in soil were less than 10 ppb.

Determination of selenium compounds in urine samples by liquid chromatography-inductively coupled plasma mass spectrometry with an ultrasonic nebulizer

An inductively coupled plasma mass spectrometer (ICP-MS) coupled with ultrasonic nebulization was used as a liquid chromatographic detector for the determination of selenium compounds. Selenite, selenate, and trimethylselenonium were separated by ion-pair reversed-phase liquid chromatography before introduction into the inductively coupled plasma. Detection limits were in the range of 22–74 pg selenium for various selenium species. The relative standard deviation of the peak heights for five injections of 10 ng ml −1 selenium mixture was in the range of 2–5%. This method has been applied to determine various selenium species in urine samples.

Determination of palladium and gold as 4,4′-bis(dimethylamino)thiobenzophenone complexes by ion-pair reversed-phase liquid chromatography

Palladium (II) and gold (III) were separated by ionpair reversed-phase, HPLC with detection at 535 nm. 4,4′-Bis(dimethylamino)thiobenzophenone (TMK) was used as a pre-column complexing agent. The optimum conditions for the separation and determination of Pd(II) and Au(III)-TMK complexes were investigated. The complexes were separated on a Nucleosil C18 column with tetrahydrofuran-water containing acetic acid-sodium acetate buffer, sodium perchlorate and TMK. The proposed method has been used to determine palladium and gold in gold recovery solution and anode mud from copper smelters.

Determination of Nitrate and Nitrite Anions in Natural Waters by Reversed-Phase Ion Pair Liquid Chromatography

The concentration of the inorganic anions nitrate and nitrite has been determined in the Guadalquivir river and the Doñana National Park located in the Southwest of Spain. Reversed-phase ion pair liquid chromatography has been employed with tetrabutylammonium bromide as the counterion in the mobile phase. Detection limits obtained for these anions are close to 0.3 ng.

Ion-pair reversed-phase liquid chromatography with fluorimetric detection of pesticides

The pesticides asulam, propoxur, coumatetralyl, biphenyl-2-ol and thiabendazole were separated by ion-pair reversed-phase liquid chromatography with fluorescent detection. The mobile phase contained methanol, acetic acid and sodium cholate as ion-pair reagent. In addition, tetramethylammonium hydrogensulphate and triethylamine were added to elute the pesticides. Separations were accomplished in less than 20 min. Interferences from nineteen pesticides were studied and recoveries from synthetic mixtures ranging from 90 to 110%. Recoveries from spiked apples and wheat grains ranged from 94 to 105% with relative standard deviations between 1.4 and 9.2% and detection limits between 0.1 and 1.9 ng.