Ion-pair Liquid Chromatographic Method Research Articles

Optimization and Validation of Reversed Phase Ion-Pair Liquid Chromatographic Method for Accurate Determination of Nitrate in High Saline Seawater

Optimization and Validation of Reversed Phase Ion-Pair Liquid Chromatographic Method for Accurate Determination of Nitrate in High Saline Seawater

Ion-pairing liquid chromatographic method determination of ketoconazole and stress degradation in pharmaceutical formulations with fluorescence detection

Ion-pairing liquid chromatographic method was validated for determination of ketoconazole in shampoo and cream samples as per ICH guidelines. The chromatographic conditions were carried out in the isocratic mode using a mixture of methanol and 8 mM sodium dodecyl sulfate (pH 5.5) in a ratio of 45:55 v/v %, as mobile phase. The flow rate was set at 1.0 mL min-1. Chromolith RP-18e (100×4.6 mm) was used as the analytical column with a fluorescence detection at an excitation wavelength of 260 nm and an emission wavelength of 375 nm. The average percentage recovery of shampoo A, shampoo B, shampoo C, cream A and cream B were 99.88, 97.06, 99.58, 96.77 and 97.26, respectively. The limit of detection was 0.12 mg L-1. The drug decomposition under acid degradation, base degradation and oxidative degradation were found to be in the range of 91.63-94.70% indicating that the drug is resistant towards acidic conditions. The drug decomposition under thermal condition and photolysis condition were found to be in the range of 69.05-87.15% and 47.31-66.83% respectively, indicating that the drug decomposition is more sensitive under photolysis conditions. This method is suitable for the quality control of ketoconazole in commercial shampoo and creams.

SEPARATION AND ANALYSIS OF AMLODIPINE/BENAZEPRIL COMBINATION IN CAPSULES BY A NOVEL ION PAIR LIQUID CHROMATOGRAPHY

Objective: The objective of this study was to develop and validate a novel ion-pair liquid chromatography method, in order to separate and assay of amlodipine/benazepril combination in capsules. This method was a fast, practical and additional choice in quality control laboratories.Methods: The chromatographic conditions comprised of a classical C18-type stationary phase (250 × 4.6 mm, 5μ), with a mobile phase consisting of: 45% of 10-3 M of cetrimide and 55% acetonitrile. The flow rate was 1 ml/min; the detection wavelength was at 242 nm, under ambient temperature.Results: The method was validated for linearity with correlation coefficients very close to one, the accuracy with mean recovery values between 95.0-105.0%, precision with relative standard deviations of the calculated concentrations less than 5.0% and specificity in the presence of degradation products and excipients.Conclusion: The results presented in this paper showed that the developed method was fast and applicable, for the separation and determination of amlodipine/benazepril combination in capsules.

Rapid ion-pair liquid chromatographic method for the determination of fenbendazole marker residue in fermented dairy products.

A simple, rapid and sensitive liquid chromatographic method that allows for the quantitative determination of fenbendazole residues in fermented dairy products is described. Samples were extracted with a mixture of acetonitrile-phosphoric acid and the extracts were defatted with hexane to be further partitioned into ethyl acetate. The organic layer was evaporated to dryness and the residue was reconstituted in mobile phase. Separation of fenbendazole and its sulphoxide, sulphone, and p-hydroxylated metabolites was carried out isocratically with a mobile phase containing both positively and negatively charged pairing ions. Overall recoveries ranged from 79.8 to 88.8%, while precision data, based on within and between days variations, suggested an overall relative standard deviation of 6.3-11.0%. The detection and quantification limits were lower than 9 and 21μg/kg, respectively. The method has been successfully applied to quantitate fenbendazole residues in Feta cheese and yoghurt made from spiked and incurred ovine milk.

Reversed-phase ion-pair liquid chromatography method for purification of duplex DNA with single base pair resolution

Obtaining quantities of highly pure duplex DNA is a bottleneck in the biophysical analysis of protein–DNA complexes. In traditional DNA purification methods, the individual cognate DNA strands are purified separately before annealing to form DNA duplexes. This approach works well for palindromic sequences, in which top and bottom strands are identical and duplex formation is typically complete. However, in cases where the DNA is non-palindromic, excess of single-stranded DNA must be removed through additional purification steps to prevent it from interfering in further experiments. Here we describe and apply a novel reversed-phase ion-pair liquid chromatography purification method for double-stranded DNA ranging in lengths from 17 to 51 bp. Both palindromic and non-palindromic DNA can be readily purified. This method has the unique ability to separate blunt double-stranded DNA from pre-attenuated (n-1, n-2, etc) synthesis products, and from DNA duplexes with single base pair overhangs. Additionally, palindromic DNA sequences with only minor differences in the central spacer sequence of the DNA can be separated, and the purified DNA is suitable for co-crystallization of protein–DNA complexes. Thus, double-stranded ion-pair liquid chromatography is a useful approach for duplex DNA purification for many applications.

Assay of Kanamycin A by HPLC with Direct UV Detection

The development of a simple reversed phase ion pair liquid chromatographic method for the assay of kanamycin A has been described. Because of the lack of a UV chromophore in the structure of kanamycin A, borate complexation was used to allow direct UV detection at 205 nm. Three columns were evaluated in this study: Zorbax Extend C18 (4.6 mm × 250 mm; 5 μm), XBridge C18 (4.6 mm × 250 mm; 5 μm) and apHera C18 (4.6 mm × 250 mm; 5 μm). The mobile phase was a mixture of 0.1 M disodium tetraborate (pH 9.0) and water (20:80, v/v) supplemented with 0.5 g L−1 sodium octanesulphonate. Final chromatographic conditions were achieved on the XBridge column at 50 °C. The method was validated according to ICH guidelines and applied to a commercially available sample. It is much faster and more specific than the current microbiological assay prescribed in the European Pharmacopoeia. No expensive equipment is necessary to perform this assay making it a viable replacement.

HPLC Stability Indicating Assay Method for Metformin Hydrochloride in Bulk Drug and Tablets and Cytotoxicity of Degradation Products

The present work reports a stability-indicating reversed phase ion pair liquid chromatography method for quantitative determination of metformin hydrochloride (MTF) (drug and tablets) and assessment of the cytotoxicity of degraded MTF. Chromatographic separation was performed on a C18 (Phenomenex® Luna), 5.0 μm (250 mm x 4.6 mm) column using isocratic elution. The optimized mobile phase consisted of 10 mmol L-1 heptane sulphonic acid (pH 3.0), acetonitrile and methanol (75:8:17 v/v), flow 1.0 mL.min-1, at 30 ºC. The eluted compounds were monitored at 210 nm wavelength using a DAD detector. The stability indicating capability of the developed method was established by analyzing forced degradation samples (acid, alkali, neutral, oxidative and photolytic condition) in which the spectral purity of MTF was ascertained, along with the separation of degradation products from the analyte peak. The cytotoxicity of MTF and the degraded drug was analyzed in the L929 cell test by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The 500 mg of commercial MTF tablets (Reference, Generic and Similar drugs) showed a similar chromatographic profile, but lower intensity degradation in the same conditions, in relation to the bulk drug in almost forced conditions. Neither the degraded drug samples, nor the tablet samples degraded in acid conditions, showed any cytotoxicity. The developed stability-indicating ion-pair HPLC method was fully validated according to the International Conference on Harmonization (ICH) guidelines. The degraded sample of MTF showed no affect on cell viability, compared with the non-degraded drug. Keywords: Bulk Drug, Cytotoxicity, Degradation Products, HPLC-UV, Ion-pair liquid chromatography, Metformin hydrochloride, Method validation, MTT assay, Stability-indicating assay method, Tablets

Impurity profiling of capreomycin using dual liquid chromatography coupled to mass spectrometry

The characterization of unknown (UNK) impurities in capreomycin (CMN) using liquid chromatography coupled to mass spectrometry (LC/MS) has been described. The ion-pair liquid chromatography method coupled to ultraviolet detection (LC–UV) described by Mallampati et al. was used for the separation of CMN from its related substances. As the method uses non-volatile reagents it could not be directly coupled to mass spectrometry (MS) for impurity characterization. So, these UNK impurities were collected and desalted before sending to MS for structural characterization. As no information about the fragmentation of the main components of CMN, except for CMN IB, was available in the literature, they were studied first. Next, the structures of the impurities were deduced by comparing their fragmentation to that of the main components of CMN. Fourteen UNK impurities that were never described before, were (partly) characterized.

Ion-Pair LC Analysis of Pyrroloquinoline Quinone in Neurotransmitter Amino Acid Incubations: Determination of Chemical Kinetics

Neurotransmitters are the chemical messengers of the brain. Many neurodegenerative diseases of the central nervous system are related to abnormal neurotransmitter activity. Pyrroloquinoline quinine (PQQ) has previously been shown to be a promising candidate for preventing cognitive deficit in neurodegeneration. To investigate whether PQQ can modulate the levels of brain neurotransmitter amino acids, a rapid and reliable ion-pair liquid chromatographic method was established and validated for the analysis of PQQ in reaction mixtures containing specific neurotransmitter amino acids. The reaction mixtures were separated on an amethyst C18-P reverse-phase column with 35:65 (v/v) acetonitrile:20 mM potassium dihydrogen phosphate, pH 5.5, containing 20 mM tetrabutyl ammonium bromide as mobile phase at a flow rate of 0.8 mL min−1. The validated method was applied successfully to study the chemical kinetics of PQQ reactions with five neurotransmitter amino acids. Order of reaction n, rate constant k, and activation energy Ea values for the reactions were calculated. This work provides important information for studying the possible protective mechanisms of PQQ in neurodegenerative diseases. Furthermore, the simplicity of this method combined with its sensitivity and reliability make it a novel contribution in the field of neurotransmitter research.

Direct separation and detection of biogenic amines by ion-pair liquid chromatography with chemiluminescent nitrogen detector

Analysis of biogenic amines is critical to pharmaceutical and food industry due to their biological importance. For many years, the determination of biogenic amines has relied on high performance liquid chromatography (HPLC) coupling with pre-, on-, or post-column derivatization procedures to enable UV or fluorescent detections. In this study, 14 biogenic amines were separated on a Phenomenex Luna ® Phenyl-Hexyl column by an ion-pair liquid chromatography method using perfluorocarboxylic acids as ion-pair reagents and detected by a chemiluminescent nitrogen detector (CLND). This direct separation and detection HPLC method eliminated the time consuming and cumbersome derivatization procedures. Compared with HPLC–UV (post-column derivatization with ninhydrin) and HPLC-charged aerosol detector (CAD) methods, this HPLC–CLND technique provided narrower peaks, better baselines, and improved separations and detections. Excellent linearity was acquired by CLND for each of the 14 biogenic amines ranging from less than 1 ng to about 1000 ng (on-column weights). The relative response factors determined by this LC–CLND method were proportional to the numbers of nitrogen atoms in each compound, which has been the characteristic of the equimolar determinations by CLND. In addition, a number of samples including beer, dairy beverage, herb tea, and vinegar were analyzed by the LC–CLND method with satisfactory precision and accuracy.

A new liquid chromatography/electrospray ionization mass spectrometry method for the analysis of underivatized aliphatic long‐chain polyamines: application to diatom‐rich sediments

Natural polyamines are found in all three domains of life and long-chain polyamines (LCPAs) play a special role in silicifying organisms such as diatoms and sponges where they are actively involved in the complex formation and nanopatterning of siliceous structures. With chain lengths extending up to 20 N-methylated propylamine repeat units, diatom LCPAs constitute the longest natural polyamines. Mixtures of natural LCPAs are typically purified in bulk using ion-exchange, size-exclusion and dialysis and then analyzed either by direct infusion mass spectrometry or by MALDI-TOF. Here, we describe a novel ion-pairing liquid chromatographic method that allows baseline separation, detection and structural elucidation of underivatized aliphatic methylated and non-methylated LCPAs with a wide range of chain lengths. Complete separation of synthetic mixtures of LCPA species differing by either a propylamine or an N-methylpropylamine unit is achievable using this method and chromatographic separation of natural, diatom frustule bound LCPAs extracted from sediment core samples is greatly improved. Using electrospray ionization mass spectrometry (ESI-MS), we detected singly [M+H](+) and multiply [M+nH](n+) charged protonated ions. The abundance of multiply charged LCPA species increased linearly as a function of LCPA chain length (N) and multiprotonated ions [M+nH](n+) were more abundant for longer chain polyamines. The abundance of multiply charged LCPAs along with the concomitant disappearance of the singly charged protonated molecular ion significantly increases the complexity of the MS spectra, justifying the need for good chromatographic separation of complex LCPA mixtures. This analytical procedure will likely constitute a powerful tool for the characterization, quantification, as well as the purification of individual LCPAs in natural and synthetic samples for studies of silica precipitation as well as nitrogen and carbon isotopic analysis used in paleoceanographic studies.

Simultaneous Ion-Pair LC Analysis of Ephedrine and Pseudoephedrine in Rat Liver Microsomal Incubations: Application to In-Vitro Metabolism

(−)-Ephedrine (ephedrine, EPH) and (+)-ephedrine (pseudoephedrine, PEPH) are metabolized by the liver, but the species of hepatocyte cytochrome P450 (CYP450) responsible is not yet clear. To investigate which subtype of CYP450 is involved in the metabolism of EPH and PEPH, a rapid and reliable reversed-phase ion-pair liquid chromatographic method for simultaneous analysis of EPH and PEPH in rat liver microsomes has been established and validated. Matrine was selected as a suitable internal standard (IS) for calibration. After liquid–liquid extraction of liver microsomal samples with methyl tert-butyl ether, EPH and PEPH were separated on a C18 reversed-phase column (200 mm × 4.6 mm, 5 μm) with methanol–0.5% sodium dodecyl sulfate–phosphoric acid–triethylamine 60:40:1.25:1 (v/v) as mobile phase at a flow rate of 1.0 mL min. Detection was by UV absorbance at 210.5 nm. For both EPH and PEPH, calibration curves were linear over the range 1.5–60.0 μg mL−1, the limit of quantification was 1.5 μg mL−1, and intra-day and inter-day variability was 73%. The validated method was successfully used to study the in-vitro metabolism of EPH and PEPH. In rat liver microsomes induced by dexamethasone, enzyme activity in the metabolism of EPH and PEPH was higher than that for metabolism of phenobarbital and β-naphthoflavone.

Reversed-phase ion-pair liquid chromatographic method for determination of reaction equilibria involving ionic species: Exemplification of the method using ligand substitution reactions of ethylenediaminetetraacetatochromium(III) ion with acetate and phosphate ions

A reversed-phase ion-pair liquid chromatographic method is presented for the determination of reaction equilibria involving ionic species of the same charge sign as reactant and product compounds. It has been demonstrated that ion-exchange chromatography or reversed-phase ion-pair chromatography is a useful tool for the determination of equilibrium constants of chemical reactions involving ionic species such as metal complexation reactions. Previous work with these methods has been based on the assumption that the limiting retention factors of the reactant and product species are constant independent of concentration of the chemical species (X) in the mobile phase, which reacts with the analyte compound. However, when all the reactant and product species are ions of the same charge sign as that of the species X, it is virtually impossible to apply these methods to the equilibrium constant determination because the retention factors of both the reactant and product species may depend on the concentration of X. In this study, an alternative approach was developed that estimates the limiting retention factors of ionic species from the dependence of the retention factor on the ionic strength of the mobile phase. Ligand substitution reactions of ethylenediaminetetraacetatochromium(III) ion with acetate and phosphate ions were used as model reactions to test this method. The equilibrium constants determined by this method are in good agreement with those obtained by a UV–visible spectrophotometric method.

Simultaneous RP-IP-HPLC assay of theophylline, phenobarbital, codeine, and ephedrine in a suppository

Summary A reversed-phase ion-pairing liquid chromatographic (RP-IP-HPLC) method for simultaneous assay of theophylline (TH), phenobarbital (PB), codeine (CD), and ephedrine (EP) in an extemporaneous (magistral) suppository was developed and validated and used to investigate a reported serious adverse event. Samples were dissolved in dichloromethane and extracted by two-step liquid-liquid extraction with acetate buffer (pH 5.0) and, subsequently, 0.1 m NaOH solution. Separation was performed on an end-capped C18 silica column with stepwise gradient elution. Sample preparation and chromatographic conditions were optimized on the basis of the pKa values of the analytes. Freedom from interference from the excipients, linearity (r2 > 0.999 for all the active ingredients), range (0.01–0.08 mg mL−1 for TH, CD, and EP; 0.0025–0.02 mg mL−1 for PB), intra-day and inter-day precision, and accuracy (recovery >95% for TH, CD, and EP and >90% for PB) of the method were demonstrated. Non-compliance of the examined produ...

CHEMICAL STABILITY OF CEFTRIAXONE BY A VALIDATED STABILITY-INDICATING LIQUID CHROMATOGRAPHIC METHOD

abstraCt The chemical stability of ceftriaxone for injection reconstituted with water was studied at different storage conditions by a validated stability-indicating ion-pair liquid chromatographic method. The study was performed on ceftriaxone for injection samples obtained from two pharmaceutical laboratories, at three different temperatures: 23 oC ± 2 oC, + 8 oC ± 1 oC and - 20 oC ± 0.5 oC, over 26 days, 55 days, and 76 days, respectively. Chromatographic separation was achieved on a RP-18 column, using a mobile phase consisting of 250 mL acetonitrile, 50 mL phosphate buffer (pH 7.4; 0.1M), 3.2 g tetrabutylammonium bromide made up to a volume of 1 L with water, at a flow rate of 1.0 mL min −1 and UV detection at 260 nm. The stability-indicating capability of the assay was established by forced degradation studies under hydrolysis, high temperature and photolysis. The developed method was specific, stability indicating, accurate and precise for ceftriaxone determination. At room temperature and at + 8 oC the degradation of ceftriaxone followed first-order kinetics and the samples were chemically stable for at least 4 and 41 days respectively. At - 20 oC the concentration of all samples remained higher than 90% of the original concentration. Degradation of ceftriaxone was higher at room temperature; therefore lower storage temperatures improved the chemical stability of ceftriaxone for injection in water.

Simple and Sensitive Determination of Metformin in Human Plasma Using an Ion-Pair LC Method

A simple, selective and sensitive ion-pair liquid chromatography method was described for the determination of metformin in human plasma. Plasma samples (200 μL) were deproteinated with 10% (v/v) perchloric acid followed by LC analysis using a Symmetry C18 column and a mobile phase of 29% acetonitrile—71% 2 mM sodium dodecyl sulfate (with 350 μL 0.1 M HCl) operated at a flow rate of 1.5 mL min−1. Metformin and the internal standard (sodium phenytoin) were detected at 232 nm and eluted at 6.8 and 7.9 min, respectively. Calibration curves were linear (r > 0.9990) between 9 and 2,000 ng mL−1. The quantitation limit was 9 ng mL−1, intra- and inter-day precision (CV) were 5.26% or less, and accuracy values were found to be within 5.95% of the nominal concentration.

An LC Ion-Pairing Method for the Determination of Fe(II) in Ferrous Bisglycinate Pharmaceutical Formulation

A reversed-phase ion-pairing liquid chromatographic method was developed and validated for the assay of Fe(II) in ferrous bisglycinate (Fe-bis-gly) capsules using 4-(2-pyridylazo) resorcinol reagent. The analysis was carried out using a Gemini RP-18 (150 mm × 4.6 mm I.D., particle size 5 μm) analytical column; the mobile phase consisted of a mixture of acetonitrile–water (28:72 v/v) containing 1 mM tetrabutylammonium hydrogensulfate and 1% phosphate buffer (pH 8.0). The flow rate was 1.0 mL min−1 and the detection was achieved with a photodiode array (PDA) detector at 706 nm. The specificity of the method was proved using stress conditions and evaluated using a PDA detector. The data validation showed that the method is specific, fast, accurate, and reproducible for the determination of Fe-bis-gly in dosage form. The response was linear over a range of 1.0–2.6 μg mL−1 (r = 0.9999). The accuracy of the method ranged from 98.02 to 102.75%. The RSD values for intra- and inter-day precision studies were below 1.3 and 1.1%, respectively. There was no interference of the excipients on the determination of the active pharmaceutical ingredient.

Liquid chromatographic determination and depletion profile of oxytetracycline in milk after repeated intramuscular administration in sheep

A simple, rapid and specific ion-pair liquid chromatographic method for the routine determination of the marker residue of oxytetracycline in sheep milk, at levels as low as 20 μg/kg, has been developed. Milk samples were acidified and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulphate and concentrated into diluted phosphoric acid. Separation was carried out isocratically on a Nucleosil C 18 column using a mobile phase that contained both positively and negatively charged pairing ions. The in-house validated method gave overall recoveries and overall relative standard deviations better than 86% and 4.6%, respectively. The method was successfully applied to study the depletion of oxytetracycline in sheep milk and to estimate the withdrawal period after intramuscular administration of a commercial oxytetracycline formulation.

Development and validation of a liquid chromatographic method for the determination of ascorbic acid, dehydroascorbic acid and acetaminophen in pharmaceuticals

A reversed-phase ion pair liquid chromatographic method (RP-LC) for the determination of dehydroascorbic acid (DHA) and ascorbic acid (AA) and also acetaminophen, which is combined in pharmaceuticals, is proposed and validated. AA and acetaminophen were analyzed directly, while DHA was determined after pre-column derivatization with 4,5-dimethyl-1,2-phenylenediamine (DMPD). The derivatization reaction was carried out under mild conditions (10 min at ambient temperature) in the dark in sodium acetate buffer (80 mM; pH 3.7) solution containing EDTA as metal scavenger. The chromatographic separations were performed on a Phenomenex Synergi 4u hydro-RP (150 mm × 4.6 mm) under isocratic elution conditions, using cetyltrimethylammonium bromide (CTAB) as ion-pairing reagent in the mobile phase. Linear responses were observed for each compound. The intra-day precision (R.S.D.) was ≤1.40% and there was no significant difference between intra- and inter-day data. Recovery studies showed good results for all compounds (99.7–101.8%) with R.S.D. ranging from 0.56 to 1.82%. The limits of quantitation were about 40, 50 and 140 pmol for acetaminophen, AA and DHA, respectively. The DHA impurity values found in dosage forms were ≤0.2% of AA.

A new liquid chromatographic method for routine determination of oxytetracycline marker residue in the edible tissues of farm animals

A simple, rapid, and specific ion-pair liquid chromatographic method for routine determination of the marker residue of oxytetracycline (OTC), namely OTC and 4-epi-oxytetracycline (4-epiOTC), in edible animal tissues (muscle, liver, kidney, and fat) has been developed. Minced tissue samples were acidified at pH 2.7 with 2 mol L(-1) sulfuric acid and extracted with acetonitrile. The extracts were purified by treatment with ammonium sulfate solution and concentrated into 0.1 mol L(-1) phosphoric acid. Baseline separation was carried out isocratically on a Nucleosil 100-5 C(18), 5-microm column using an acetonitrile-0.01 mol L(-1) disodium hydrogen phosphate (20:80, v/v) mobile phase that contained both positively (tetrabutylammonium) and negatively (octanesulfonate) charged pairing ions and EDTA, and was adjusted to pH 3.8. Detection was by UV at 370 nm. The method was fully validated according to Commission Decision 2002/657/EC. Overall recoveries were better than 82.6% and overall relative standard deviation was better than 6% for all the tissues examined. The good analytical characteristics of the method allowed limits of quantification as low as 30 ng g(-1) for muscle and fat and 50 ng g(-1) for liver and kidney, for both OTC and 4-epiOTC, to be realized. The method was successfully used to determine the OTC marker residue in tissues of two sheep intramuscularly administered a commercial OTC formulation.