Ion-pair Technique Research Articles

Determination of astemizole, terfenadine and flunarizine hydrochloride by ternary complex formation with eosin and lead(II)

A simple and sensitive spectrophotometric method has been established for the determination of astemizole(I), terfenadine(II) and flunarizine hydrochloride(III) based on ternary complex formation with eosin and lead(II). The method does not involve solvent extraction. The colour of the produced complex is measured at 547.5 nm for (I) and (III), while (II) is measured at 540.7 nm. Appropriate conditions were established for the colour reaction and for the eosin: Pb(II): drug ratio to obtain maximum sensitivity. Under the proposed conditions, the method is applicable over concentration range of 4.1–37.6, 11.8–47.2 and 2.4–19.1 μg ml −1 with mean percentage recovery of 99.20±0.63, 99.76±0.39 and 99.60±0.47% for (I), (II) and (III), respectively. The suggested method was applied for determination of (I), (II) and (III) in pharmaceutical preparations. Through the use of a non-ionic surfactant (methylcellulose), prior extraction of the drugs was unnecessary. The results obtained demonstrated that the method is equally accurate, precise and reproducible as the official or reported methods. For the purpose of enhancing the sensitivity, a fluorescence quenching method for determination of the studied drugs via ternary complex formation was also investigated. The detection limit for the studied drugs (I), (II) and (III) was 0.94–7.1 μg ml −1 with mean percentage recovery of 99.84±0.29, 99.24±0.36 and 99.34±0.26%, respectively. The results obtained by applying the proposed methods were statistically analyzed and compared with those obtained by official or reference methods. Unlike other reported ion-pair techniques, the suggested methods have the advantage of being applicable for the determination of the three drugs in their pharmaceutical dosage forms without prior extraction. They are recommended for quality control and routine analysis where time, cost effectiveness and high specificity of analytical techniques are of great importance.

Highly polar metabolites of nitroaromatic compounds in ammunition wastewater

Transformation processes of nitroaromatic compounds (NAC) are described that lead to polar and highly hydrophilic metabolites in ammunition waste water. For the identification of proposed metabolites, several analytical methods for the separation and determination of these compounds were developed and applied to the investigation of a former ammunition plant and its surroundings. The compound classes which were investigated include nitro and amino substituted toluenesulfonic acids, nitrobenzoic acids, aminobenzoic acids and nitromethylphenols. Method development was usually done with high performance liquid chromatography and ion pairing or ion suppression techniques. In the leachate water of a disposal site of a former ammunition plant in Stadtallendorf, Germany, 15 compounds from the above-mentioned categories have been found to be present in concentrations up to a few hundred μg/L. The presence of so many polar compounds suggest that current and future surveying programs should include representative analytes of the investigated categories.

Determination of Acidic Pesticides in Water by a Benchtop Electrospray Liquid Chromatography Mass Spectrometer

A very sensitive and specific analytical procedure for determining 20 acidic pesticides in aqueous environmental samples using pneumatically assisted electrospray (ES) LC/MS is presented. This procedure involves passing 1- and 4-L river water and drinking water samples, respectively, through a 1-g graphitized carbon black (GCB) extraction cartridge. By exploiting the presence of positively charged active centers on the GCB surface, isolation of the acidic pesticides from base/neutral species was made possible by differential elution. Recoveries of the analytes were higher than 85%, irrespective of the aqueous matrix in which they were dissolved. Adoption of the ion pair technique by addition of 0.1 mmoVL K 2 HPO 4 and 0.2 mmoVL tetrabutylammonium fluoride to the mobile phase allowed analytes to be analyzed as preformed ions. A conventional 4.6-mm-i.d. reversed-phase LC C-18 column operating with a 1 mL/min flow of the mobile phase was used for chromatographing the analytes. A flow of 30 μL/min of the column effluent was diverted to the ES source, while the rest of the mobile phase was delivered to a UV detector set at 220-nm wavelength. The effects of the nature of the ion pair forming agent and its counterion as well as its concentration in the mobile phase on the response of the ES/MS detector were investigated. The effects of varying the skimmer cone voltage on the production of diagnostic fragments and the response of the MS detector were also evaluated. For the analytes considered, the response of the mass detector was linearly related to the amount of the analytes injected between 2.5 and 200 ng. A certain variation of the ion signal for the analytes considered occurred after several hours of continuous use of the LC/ES/MS instrumentation. For routine use, then, analyte quantitation could be better performed by the UV trace method while entrusting unambiguous identification of the analytes to the MS detector. The limits of sensitivity (signal-to-noise ratio = 3) of the method for the pesticides considered in drinking water and surface water samples were estimated to be about 2-6 and 8-25 ng/L, respectively.

Alkylsulphonic acid ion pairing with radial compression columns for determining plasma or cerebrospinal fluid 1-β- d-arabinofuranosylcytosine in pediatric pharmacokinetic analysis

In order to accurately and precisely measure plasma and cerebrospinal fluid(CSF) 1-β- d-arabinofuranosylcytosine (Ara-C) in pediatric samples with adequate sensitivity and without interference, we have developed a reversed-phase ion-pairing technique utilizing the free amino group of the pyrimidine ring of Ara-C. Optimum resolution and separation was achieved utilizing a 4 μm C 18 radial compression column. Ara-C and the internal standard 8-bromo-cyclic-AMP eluted at 6.5 and 4.6 min, respectively, with complete resolution. The minimum detectable amount is 2.5 pmol in a 50-μl volume. The assay was linear in both plasma and CSF. Intra- and inter-day assay precision were less than 4% and 9%, respectively, for plasma with similar results obtained for CSF. Neither endogenous compounds nor commonly co-administered drugs interfere. Validity for our method was supported by the successful assay of over 400 pediatric plasma and 50 CSF samples for pharmacokinetic analysis. The method offers accuracy, precision, sensitivity and efficiency for plasma or CSF Ara-C determination.

Ion-pair reversed-phase liquid chromatographic determination of dihydralazine

Dihydralazine, a vasodilator drug chemically related to the structural class of the hydrazinophthalazines, was used as a tracer to monitor (water-oil-water) multiple emulsion stability. The hydrophilic character of the ionized form of dihydralazine hinders its Chromatographic behaviour by inducing a low retention with alkyl-grafted supports. In addition, the two hydrazine functions induce some important interactions with the polar sites of the stationary phase. This yields highly tailing and poorly retained peaks, hence there is a need to develop an ion-pair technique on a stationary phase recommended for the analysis of basic drugs. After optimization, the optimum chromatogram was obtained with a C 8 RP-Select B column using a mobile phase consisting of a mixture of methanol (50%) and an aqueous solution of phosphate (2.5 · 10 −2 M) and sodium heptanesulfonate (2.5 · 10 −2 M) at pH 4.4. This method was found to be accurate and precise for a range of dihydralazine concentrations from 1 to 120 μg ml .

Redox Chemistry and [Au (CN)2 -] in the Formation of Gold Metabolites

The role of hypochlorite ion, which can be generated by the enzyme myleoperoxidase, in the biochemistry of gold(I) anti-arthritic drugs was investigated. Sodium hypochlorite (OCl−) directly and rapidly oxidizes AuSTm, Au(CN)2-, AuSTg (gold thioglucose) and auranofin (Et3PAuSATg). The resulting gold(III) species were detected by an Ion Chromotography Ion-Pairing technique that was developed to distinguish gold(I) and gold(III). Formation of Au(III) was also demonstrated spectrophotometrically after the conversion to AuCl4−. The reactions of AuSTm, AuSTg, and auranofin are complex and gold(III) appears only after the initial oxidation of the thiolate (and phosphine) ligands. The enzymatic reaction, using MPO with H2O2 and Cl− as substrates, leads to slow oxidation of Au(CN)2-, AuSTm or AuSTg. The extent and rate of reaction depend on the concentrations of MPO, H2O2, and Au(I). The continued presence of Au(I) during the initial stages of reaction (oxidation of the thiolates in AuSTm and AuSTg) and the conversion to Au(III) in the latter stages of the reaction were demonstrated. Au(CN)2-, a gold metabolite, binds tightly to serum albumin. Unlike other gold(I) complexes, aurocyanide reacts almost negligibly at Cys-34 via ligand exchange. Instead, there is a strong association (K1 = 5.5 × 104 and K2 = 7.0 × 103; n1 = 0.8 and n2 = 3) of intact Au(CN)2-. The full extent of binding is revealed only by equilibrium methods such as NMR or ultrafiltration; the bound gold dissociates extensively on conventional gel-exclusion columns and partially on Penefesky spun columns. The immunological and pharmacological significance of these results are discussed.

A rapid and sensitive determination of salmon calcitonin in solutions containing bovine serum albumin or gelatin.

A sensitive and selective reversed phase high performance liquid chromatographic method was introduced for the determination of salmon calcitonin (sCT) in solutions containing bovine serum albumin or gelatin. The method was based on the sensitive fluorescence detection of sCT by postcolumn derivatization with o-phthal(di)aldehyde. The sample was loaded onto a reversed phase column, purified by column switching and separated with linear gradient elution using the ion pair technique. A linear relationship was obtained between the peak height and the amount injected in the concentration range 1-40 ng. The detection limit was 10 ng/mL (S/N = 3) at a sample injection volume of 100 microL.

Reversed-phase high-performance liquid chromatographic determination of linear alkylbenzene sulphonates, nonylphenol polyethoxylates and their carboxylic biotransformation products

The possibility was investigated of simultaneously determining linear alkylbenzene sulphonates, nonylphenol polyethoxylates and their respective biotransformation products, namely sulphophenyl carboxylates (SPC) and nonylphenoxy carboxylates, by reversed-phase high-performance liquid chromatography with UV and fluorescence detection. Both the ion-suppression and the ion-pair techniques were taken into account for separating the compounds of interest. Each technique exhibited peculiar characteristics of resolution and sensitivity. Differences in the selectivity of the chromatographic system on using either methanol or acetonitrile as organic modifier were also considered. Liquid-solid extraction by an octadecyl-bonded silica (C 18) cartridge was employed to isolate all the compounds considered from a sewage treatment plant effluent sample. The recovery of SPC with up to six carbons in the carboxylate chain was unsatisfactory. The SPC distribution appeared to be dominated by the homologues having 5–8 carbon atoms in the carboxylate chain.

A Simple Method for the Separation of Triterpene Carboxylic Acids

Abstract A polybutadiene-coated alumina column has been found to resolve a closely related mixture of naturally occurring triterpene carbox-ylic acids much more efficiently than conventional octadecylsilyl-bonded (C18) silica columns. Further, this separation could be achieved without ion-pairing, ion-suppression or other buffering techniques.

Separation of aminoacidato ruthenium(II) complexes by ion-pair chromatography

The chromatographic conditions for analytical separation of (η6-C6H6) ruthenium(II) complexes with amino acid and dipeptide ligands have been investigated in dependence on their molecular structure. Different chromatographic phases were tested and the efficiency parameters were determined by the reversed-phase ion-pair technique. For the enantiomeric species L-penicillaminato ruthenium(II) complexes cis- and trans-[(η6-C6H6)Ru(L-Pen)H−1]2Cl2 and the reaction byproducts a complete chromatographic separation has been obtained using sodium dodecylsulphate as an ion-pair reagent. For the purification procedure of the L-penicillaminato ruthenium(II) complexes, in order to characterize the separated pure complexes by 1H-NMR spectroscopy, a preparative separation has been carried out using the volatile trifluoroacetic acid as an ion-pair reagent.

Solid-phase extraction and HPLC-UV confirmation of drugs of abuse in urine.

A series of six liquid chromatographic methods were developed to confirm the presence of six classes of drugs of abuse in urine. The chromatographic separations were performed with a reversed-phase C8 column, except in the case of morphine, which was separated on a normal phase column. Isocratic and gradient elutions, ion pair technique, and UV detection were employed. Sample pretreatment involved the extensive application of solid-phase extractions and liquid-liquid extractions on solid supports. The specificity and sensitivity enabled the confirmation of morphine, benzoylecgonine, THC-COOH, amphetamine, and methamphetamine, six barbiturates, and nine benzodiazepines screened positive by EMIT in urine.

Application of ion pair high performance liquid chromatography to the analysis of porphyrins in clinical samples

Reversed phase ion pair chromatography is a highly selective separation technique for the determination of free porphyrin carboxylic acids from human materials. Isocratic and gradient elution methods can be used to analyse porphyrin isomers and to establish porphyrin profiles for the biochemical diagnosis of porphyrias. Ion pair high performance liquid chromatography led to the discovery of the atypical isomers II and IV of uroporphyrin and coproporphyrin in human urine. Advantages and limitations of the ion pair technique are discussed.

Plasma ochratoxin A levels in three Swedish populations surveyed using an ion‐pair HPLC technique

A new HPLC method for the analysis of ochratoxin A in plasma samples is described. The analysis is performed at an alkaline pH using an ion-pair technique, fluorescence detection at an excitation wavelength 380 nm, and an emission wavelength 420 nm. The detection and quantification limits are 0.02 ng and 0.05 ng ochratoxin A/ml plasma, respectively. The method was used to determine the ochratoxin A content of human plasma samples, collected in three districts of Sweden. The Visby district had a significantly higher proportion of ochratoxin A positive samples and higher levels than the other two districts--Uppsala and Ostersund. The calculated daily intake of ochratoxin A in the Visby district (0.35 ng/kg body weight), exceeds the lower tolerable daily intake (TDI) value suggested by Kuiper-Goodman and Scott (1989). The calculated daily intake by the population on the mainland of Sweden (0.04 ng/kg body weight) is below the proposed TDIs.

Determination of total glycyrrhetic acid in Glycyrrhizae Radix by second derivative UV spectrometry

Second derivative (D2) spectrometry using ion-pair extraction technique was developed for the determination of total glycyrrhetic acid (GA) in Glycyrrhizae Radix. Glycyrrhizin (G) obtained from Glycyrrhizae Radix was hydrolyzed into GA in 2 N-HCl and methanol (1∶1) and extracted from aqueous phase in the form of an ion-pair complex with tetrapentylammonium bromide (TPA) as a counter ion. Maximum D2 amplitude (Z value) was obrained when 1000-fold or greater molar ratio of TPA was used at pH 11. Reaction time, temerature and ionic strength did not affect ion-pair formation. Dichloromethane was an effective extraction solvent of the ion-pair complex. The linearity of standard curve of ion-pair GA was obtained in the range of 4–120 μg/ml as GA. Assayed contents of GA in dry powder by D2 UV spectrometry and HPLC method were 5.31±0.04% and 5.20±0.008%, respectively.

Separation of ionic drug substances by supercritical fluid chromatography

The use of ion-pairing principles in supercritical fluid chromatography (SFC) is demonstrated. Using this technique, the scope of SFC can be extended to include ionic and ionizable compounds. The possibilities of ion-pair techniques and the advantages of SFC over high-performance liquid chromatography (HPLC) are discussed. The influence of different ion-pairing reagents on the selectivity was elucidated for a wide range of pharmaceutical compounds. The dependence of efficiency and selectivity on the eluent density was investigated, leading to an optimization strategy for cationic and anionic substances including matching ion-pair reagents, the stationary phase and density programming. It was demonstrated that owing to the combined influences on selectivity of the ion-pair type and the density, together with the high efficiency of SFC, optimized separations are obtained faster by SFC than by HPLC. The limitations of ion-pair SFC are mainly due to the solubility of ion-pairing reagents in the carbon dioxide-modifier mixtures. The solubility of typical ion-pairing reagents under different SFC conditions was investigated.

Analysis and Stability of Orotic Acid in Milk

A method based on HPLC and ion pairing techniques was used to determine the orotic acid concentrations in cows’ milk. Average amounts were 69 to 74 mg/L. Orotic acid in milk was stable to heating but decreased during commercial yogurt fermentation. Correlations were made between the loss of orotic acid and the increase or decrease in pH during fermentation.

Simultaneous determination of biogenic amines, their precursors and metabolites in a single brain of the cricket using high-performance liquid chromatography with amperometric detection.

An analytical procedure has been developed for the simultaneous determination of biogenic amines, their precursors and metabolites by high-performance liquid chromatography with amperometric electrochemical detection. Following careful adjustment of various factors involved in the separation efficiency, reversed-phase chromatography with an ion-pairing technique gave simultaneous separation of nineteen biogenic amines and related substances. Peak identification was confirmed by comparison with hydrodynamic voltammograms. The method was sensitive enough to detect each substance in the picomole range. The procedure was applied to quantitate the amount of biogenic amines in a single brain of the cricket.

High-performance liquid chromatographic technique for the determination of the polyether antibiotic lysocellin sodium

A high-performance liquid chromatographic technique is described for the polyether antibiotic lysocellin sodium. The technique was developed because the methods currently used for polyether antibiotics are not applicable to lysocellin sodium. This is because lysocellin sodium either lacks a chromophore, does not react with vanillin or lacks the stability needed for derivatisation. The proposed technique involves the use of a UV-absorbing ion pair and is rapid and specific for lysocellin sodium. A method for the determination of lysocellin sodium in fermentation broth which uses the UV-absorbing ion-pair technique is discussed.

Simultaneous ion-pair and partition liquid chromatography of acetaminophen, theophylline and salicylate with application to 500 toxicologic specimens.

Acetaminophen (A), salicylate (S), and theophylline (T) are three common pharmaceutical agents involved in drug overdose for which determinations of serum drug concentrations play a pivotal role in clinical management. All three must be included in overdose screening, but often separate methods must be used. By combining reverse phase high performance liquid chromatography (HPLC) for A and T with ion-pair techniques for S, all three drugs can be chromatographed and quantitated simultaneously. A water-soluble ion-pair of S and 2-amino-2-(hydroxy-methyl)-1,3-propanediol eluted in a mobile phase of 4% acetonitrile and 12% acetic acid in water. Relative retention times for A, T and S were 0.41, 0.57 and 1.75 with an internal standard of 8-chlorotheophylline. Validation studies demonstrated recoveries of 88-104%, intraasay precisions of 2.1-6.6%, interassay precision of 3.8-8.0%, detection limits and linearity ranges of 2-200 mg/l for A and T and 10-1,000 for S. Seventy-two drugs were tested for interference. Accuracy studies showed no significant biases when compared to other methods. The method is convenient for toxicologic screening or therapeutic drug monitoring. In 576 consecutive serum specimens submitted for general toxicologic screening, 7.3% were positive for A, 5.2% for S, and 1.7% for T. Only 1.7% had critical concentrations indicating intoxication.

Liquid chromatographic determination of the pH-dependent degradation of eseroline — hydrolysis product of physostigmine

The stability of eseroline, the hydrolysis product of physostigmine under aerobic conditions, was studied by liquid chromatography. A reversed-phase, ion-pair technique was used to separate eseroline from its degradation products. The degradation of eseroline in phosphate buffer solutions of pH 6.91, 7.40, 7.98, 8.41 and 8.94 appears to follow first-order kinetics; the rate constant increased with an increase in pH. The degradation appears to follow specific base catalysis.