HPLC Method For Separation Research Articles

Análise, por CLAE, de carotenóides de cinco linhagens de Rhodotorula

Um método por cromatografia líquida de alta eficiência (CLAE) foi otimizado para a análise da composição de carotenóides de cinco linhagens de Rhodotorula.A extração com ruptura mecânica da parede celular da levedura com areia tratada mostrou ser mais eficiente que a ruptura química com dimetilsulfóxido. Os carotenóides foram separados e quantificados por CLAE em coluna de C18 utilizando como fase móvel acetonitrila/metanol (0,1% trietilamina)/acetato de etila (75:15:10) e 100% metanol (0,1% trietilamina) entre as injeções, com vazão de 1 mL/min. Em todas as linhagens, os carotenóides majoritários encontrados foram torularrodina, toruleno, ³-caroteno e ²-caroteno. Os teores totais de carotenóides, em µg/g, foram de 251,7 em R. glutinis,123,5 em R. rubra,113,2 em R. araucariae,105,8 em R. lactosa ede 103,7 em R. minuta.

Development of a simple HPLC method for separation of doxycycline and its degradation products

A simple HPLC method for the separation of doxycycline and its degradation products 6-epidoxycycline and metacycline was developed. Numerous HPLC conditions were tested for the qualitative determination of doxycycline and its degradation products. The best result was achieved by using Phenomenex® Luna 5 μm C 8 250×4.6 mm column with a Phenomenex® C 8 4×10 mm I.D. guard column, and a mobile phase consisting of acetonitrile:water:perchloric acid (HClO 4) (26:74:0.25) adjusted to pH 2.5 with 5 M sodium hydroxide, a flow-rate of 1.0 ml/min and ultraviolet detection at 350 nm. Correlation coefficients for calibration curves within the detection range of 3–60 μl/ml were 0.9990 for doxycycline and 1.000 and 0.9994 for 6-epidoxycycline and metacycline, respectively (within the range 0.5–7 μl/ml). The resolution between metacycline and 6-epidoxycycline was 1.2 and between 6-epidoxycycline and doxycycline it was 1.9 which fulfils European Pharmacopoeia requirements. The within- and between-day precision was determined for both retention time and peak area. Preliminary results indicate that this method can also be applied for separating other tetracyclines such as minocycline, chlortetracycline, tetracycline and demeclocycline.

Ion‐pairing high‐performance liquid chromatography‐mass spectrometry of impurities and reduction products of sulphonated azodyes

AbstractAn HPLC separation method with triethylammonium acetate mobile phase additive developed for the analysis of impurities in polysulphonated azo dyes provides good separation selectivity and compatibility with electrospray ionisation (ESI) mass spectrometry. The negative‐ion ESI mass spectra containing only peaks of deprotonated molecules [M–H]– for monosulphonic acids, [M–xH]x–, and sodiated adducts [M–(x + y)H + yNa]x– for polysulphonic acids allow easy molecular mass determination of unknown impurities. Based on the knowledge of the molecular masses and of the fragment ions in the MS/MS spectra, probable structures of trace impurities in commercial dye samples are proposed. To assist in the interpretation of the mass spectra of complex polysulphonated azodyes, additional information can be obtained after chemical reduction of azodyes to aromatic amines. The structures of the non‐sulphonated reduction products can be determined by reversed‐phase HPLC/MS with positive‐ion atmospheric pressure chemical ionisation and of the sulphonated products by ion‐pairing HPLC/MS with negative‐ion ESI.

New HPLC method for separation of blood plasma phospholipids

The aim of the present work was to develop a new HPLC method for separation of phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI) and lysophosphatidylcholine (LPC) from small-volume samples of blood plasma. Human plasma glycerophospholipids were separated by liquid–liquid extraction method followed by solid phase extraction (SPE) on aminopropyl columns. Reversed-phase Sephasil C8 column (10 cm×2.1 mm, I.D. 5 μm) and micropreparative chromatograph “SMART” were used for separation of PC, PE, LPC and PI from SPE phospholipids extract. Binary-step gradient of eluent A: acetonitrile–methanol (130:5, v/v) and B (0.01% trifluoroacetic acid) provided good, fast and reproducible resolution of investigated phospholipids classes in 12 min at 30 °C. Eluted phospholipids were detected at wavelengths λ=235 and 254 nm. This method made it possible to determine quantitatively: 5 μg ml −1 PC, 1 μg ml −1 LPC, 4 μg ml −1 PE and 3 μg ml −1 PI in blood plasma samples.

Reversed-phase HPLC determination of mangiferin, isomangiferin and hesperidin in Cyclopia and the effect of harvesting date on the phenolic composition of C. genistoides

A reversed-phase HPLC method for separation of polyphenols in honeybush tea (Cyclopia spp.) is presented. Separation of eriodictyol, luteolin, medicagol, formononetin, mangiferin, isomangiferin, hesperetin and hesperidin was investigated. A C12 stationary phase was required to separate mangiferin and isomangiferin. The method was used to quantify the three major polyphenols (mangiferin, isomangiferin and hesperidin) in C. genistoides, C. intermedia, C. maculata and C. sessiliflora and to study the effect of harvesting date on these compounds in two types of C. genistoides. The highest levels of the xanthones, mangiferin (3.61 g/100 g) and isomangiferin (0.54 g/100 g), and the flavanone, hesperidin (1.74 g/100 g), were found for C. genistoides (both xanthones) and C. intermedia, respectively. Cyclopia sessiliflora contained the lowest levels of mangiferin (1.04 g/100 g) and hesperidin (0.29 g/100 g). The mangiferin content of both the Overberg and West Coast types decreased with harvesting date (P <0.05). The Overberg type contained more mangiferin, but hesperidin was more prominent in the West Coast type.

Novel Structure of Fullerenes and Endohedral Fullerenes

AbstractBy using Krätschmer-Huffman synthesis and HPLC separation method we have isolated a series of novel fullerenes and endohedral fullerenes. Various spectroscopic techniques, e.g., MS, NMR, UV-vis-NIR, TEM, X-ray diffraction spectrometry etc., were adopted to characterize the isolated fullerenes. Several fullerenes were revealed to hold novel structures and electronic properties. For examples, C80(D5d) was isolated and characterized to have an ellipsoidal structure which is in fact one of the shortest SW-nanotubes; The isolation of Sc2@C66 breaks the well-known isolated-pentagon-rule (IPR) for the first time, which shows that the unconventional fullerenes may be dramatically stabilized through encaging metal atoms; Sc2C2@C84 is a novel molecular endohedral fullerene in which the Sc2C2 cluster rotates rapidly along the main C2 axis of C84(D2d). This fullerene is predicted to be a molecular magnet and may be used as nano-switcher in electronics.

HPLC isolation of the anti-plasmodially active bisbenzylisoquinone alkaloids present in roots of Cissampelos mucronata.

The methanolic extract of dried, powdered Cissampelos mucronata roots possesses significant in vitro activity against Plasmodium falciparum. In order to enable further pharmacological testing, the substances responsible for the observed activity were purified, mainly by HPLC, using various stationary and mobile phases. The active principles were determined to be a series of bisbenzylisoquinoline alkaloids, a group of natural products for which one of the first routine preparative HPLC separation methods is described.

Intelligent automation of high-performance liquid chromatography method development by means of a real-time knowledge-based approach

We describe the development, attributes and capabilities of a novel type of artificial intelligence system, called LabExpert, for automation of HPLC method development. Unlike other computerised method development systems, LabExpert operates in real-time, using an artificial intelligence system and design engine to provide experimental decision outcomes relevant to the optimisation of complex separations as well as the control of the instrumentation, column selection, mobile phase choice and other experimental parameters. LabExpert manages every input parameter to a HPLC data station and evaluates each output parameter of the HPLC data station in real-time as part of its decision process. Based on a combination of inherent and user-defined evaluation criteria, the artificial intelligence system programs use a reasoning process, applying chromatographic principles and acquired experimental observations to iteratively provide a regime for a priori development of an acceptable HPLC separation method. Because remote monitoring and control are also functions of LabExpert, the system allows full-time utilisation of analytical instrumentation and associated laboratory resources. Based on our experience with LabExpert with a wide range of analyte mixtures, this artificial intelligence system consistently identified in a similar or faster time-frame preferred sets of analytical conditions that are equal in resolution, efficiency and throughput to those empirically determined by highly experienced chromatographic scientists. An illustrative example, demonstrating the potential of LabExpert in the process of method development of drug substances, is provided.

HPLC/UV determination of organic acids in fruit juices and nectars

A reversed phase HPLC method for separation and determination of organic acids in fruit juices and nectars is presented. The method is based on the reaction of free organic acids with O-(4 nitrobenzyl)-N,N′-diisopropylisourea (PNBDI) in presence of dioxane. Excess of reagent was removed with a strong cation-exchange resin. The p-nitrobenzyl esters were separated on a C18 reversed phase column using gradient elution with water and acetonitrile and UV detection at 265 nm. Benzylmalonic acid was used as internal standard. The determinations were performed in the linear range of 0.05–2 g/l for lactic, acetic and succinic acids, 0.1–12 g/l for tartaric, malic and citric acids. The detection limits were 0.025 g/l, 0.017 g/l, 0.050 g/l, 0.039 g/l, 0.025 g/l and 0.060 g/l, respectively for lactic, acetic, tartaric, malic, succinic and citric acids. Validation of the method was carried out by the standard additions method, the recoveries ranged from 91.4 to 103.0%. The precision of the method was also evaluated and reported a CV% as less than 3.1%. Organic acid profiles of citrus fruit, pineapple and apple natural juices (home-made), commercial juices and nectars were established.

Phenolic compounds in virgin olive oil. 2. Reappraisal of the extraction, HPLC separation, and quantification procedures.

The extraction procedures (solid/liquid SPE and liquid/liquid LLE) and HPLC separation and quantification methods of polyphenolic compounds have been checked in virgin olive oils in order to explain the differences in content reported in the literature. The work has been carried out on oils prepared from one cultivar and produced under the same protocol. The extraction methods are practically equivalent, but the SPE technique is more favorable because it is faster and simpler. It has been proved that the chromatographic features and the method of chemical expression of the concentrations may greatly affect the final values. Thus, under the same analytical method, the total concentration values of polyphenols of the same oil show variations from 18% to 80%, according to the formality of expression as gallic acid, caffeic acid, or tyrosol equivalents. The role of the nature and spectrophotometric features of the phenols and of the internal standard is also discussed, and it was found to be an important source of reported variation. A gradient separation with an eluent mixture acetonitrile-sulfuric acid (0.1 mol/L), detection at 225 nm, and quantitative calculation of polyphenolic compounds in oils (expressed as tyrosol equivalents, THY(eq)) is proposed.

Selective Detemination Method for Vanadium (V) and Vanadium (IV) Controlling the pH of Media for a Solid-Liquid Extraction Column.

Solid-liquid extraction separation based on a speciation method was studied for selective determination of vanadium (IV) and vanadium (V). Both V (IV) and V (V) cations transform to oxo-acid anion along with pH changes in the solution. The pH values for the transition points are different from each other and the difference was utilized in the separation. In the first step, particulate samples are dissolved by strong acids such as 1 M H2SO4 or 85% H3PO4. The pH of the strong acidic sample solutions of V (IV) and V (IV) are adjusted to the range between 3 and 4. In this condition, V (IV) is in the form of cation but V (V) is anion. The pH adjusted solution is applied to an anion exchange column. The solution is expected to contain V (IV) only. The trapped V (V) anion is eluted as VO2+ cation by a pH 1 acid. The author and coworker have already developed an HPLC separation method utilizing this separation concept. However, the HPLC method has some limitations, mainly originated in physical and chemical weaknesses of the HPLC column. In the present study, a firm solid-liquid column is adopted to replace the feeble HPLC column as a separation device. And a simple and convenient pH adjustment technique for making the sample solution is investigated at the same time. With these improvements, the speciation method developed with strong acidic solutions could determine the amount of V (IV) and V (V) in various environmental and biological samples.

Stability and compatibility of an aerosol mixture including N-acetylcysteine, netilmicin and betamethasone

The physicochemical stability and the compatibility between N-acetylcysteine (1 g/5 ml), betamethasone (4 mg/1 ml) and netilmicin (100 mg/1 ml) were studied at room temperature (25±2°C) over 1 h. During this study, drug concentrations were measured using three separate HPLC methods with UV detection at t=0, 5, 10, 20, 30, and 60 min. The pH of the mixture was determined. Degradation products of the drugs were assayed using HPLC. This study demonstrates the stability and compatibility of the mixture over 1 h at room temperature. The pinkish non-remnant coloration observed when pouring N-acetylcysteine into a recipient has no effect on the stability of the drug.

Detection of carbohydrates by electrospray ionization-ion mobility spectrometry following microbore high-performance liquid chromatography

This paper reports the first example of electrospray ion mobility spectrometry as a detection method for HPLC separation, demonstrating its potential for the quantitative and selective detection of non-volatile and non-chromophoric organic compounds. Reduced mobility constants ( K 0) for 21 carbohydrates, including simple sugars, sugar alcohols and amino sugars, were determined to range from 0.68 to 1.37 cm 2 V −1 s −1. Minimum detectable quantities were measured from as low as 5.8·10 −14 mol for d(+)-cellobiose to as high as 8.2·10 −11 mol for l-iditol. A plot of carbohydrate mass with respect to inverse ion mobility indicated that, to a first approximation, mobility was inversely proportional to mass for most carbohydrates. However, in several cases, isomeric separation could be achieved by ion mobility spectrometry, demonstrating that ion mobility separation is based on size and shape of the molecule rather than molecular mass. Other notable exceptions to the mobility-mass relation were ß- d-maltose and d(+)-cellobiose, which exhibited significantly lower mobilities than expected, and maltoheptaose, which exhibited a significantly higher mobility than expected. Dimerization and multiple charges may have caused these deviations from the simple mass-mobility correlation. Parametric optimization coupled with further basic investigations of this ionization and detection method should lead to a novel approach for the analytical determination of carbohydrates.

Transition-metal complexes as alternatives to proteolytic enzymes. Regioselective cleavage of myoglobin by palladium(II) aqua complexes

The palladium(II) aqua complexes [Pd(H2O)4]2+, cis-[Pd(en)(H2O)2]2+, and cis-[Pd(dtco-OH)(H2O)2]2+ effect hydrolytic cleavage of horse myoglobin in aqueous solution. The conditions were optimized with the third complex. Its structure was determined by X-ray crystallographic analysis of its precursor, the square-planar complex cis-[Pd(dtco-OH)Cl2], in which the chelating ligand adopts a boat-chair conformation. A weak interaction between the hydroxyl group and the palladium(II) atom seems to improve the stability of the reagent. The yield of cleavage after a 24-h incubation at 60 °C increases from 39% to 85% as the pH decreases from 6.2 to 3.2. The protein fragments are separated by SDS-PAGE electrophoresis and HPLC separation methods, and identified by ESIMS and MALDI-TOF mass spectrometric methods and by determination of terminal amino-acid sequences. Most of the 13 cleavage sites are clustered around the methionine, arginine, and some of the histidine residues, whose side chains can bind to palladium(II). Cleavage tends to occur at the peptide bonds one to three positions removed from the binding residues; the scissile bonds usually lie on the amino-terminal side, seldom on the carboxy-terminal side, of the binding residues. Removal of the heme and unfolding of the protein do not drastically alter the pattern of cleavage. The ability of palladium(II) aqua complexes to cleave proteins at relatively few sites, with explicable selectivity, with good to very good yield, and in weakly acidic and nearly neutral solutions, bodes well for their future use in biochemical and bioanalytical practice.

Stability of metallofullerenes following neutron capture reaction on the metal ion

The stability of 16 kinds of metallofullerenes (9 elements) was investigated against the recoil phenomena associated with the capture of neutrons by the metal atoms inside the carbon cages. The yields of the unaffected species were obtained by use of the HPLC separation method and radioactivity measurements. Recoil energy distributions of the metal atoms were calculated theoretically and the effective energy required for destruction of metallofullerenes from inside by recoiling atoms was estimated to be about 10 to 15 eV for the metallofullerenes containing lanthanide atoms.

Automated sample preparation for drugs in plasma using a solid-phase extraction workstation

An automated solid-phase extraction workstation was used to develop, characterize and validate two separate HPLC methods for quantifying drugs in plasma. Method development was facilitated by workstation functions which allowed wash solvents of varying organic composition to be mixed and tested automatically. The precision estimates for the two methods were within 6.0 and 2.0% RSD across their respective calibration ranges. Accuracies for replicate determinations of quality controls were between −1.2 and +4.8% RE over ng ml −1 and μg ml −1 calibration ranges, respectively. Optimized recoveries were quantitative and were generally greater than 90% for the four analytes tested, and depended to a great extent, as expected, on the composition of the wash solvent. Sample throughput benchmarks for the two methods ranged from 3 to 10 min per sample, depending on the extent of air drying used. Because of parallel sample processing, 60 samples could be extracted in as little as 17 min.

Stability and compatibility of topotecan hydrochloride for injection with common infusion solutions and containers

The stability and compatibility of topotecan hydrochloride with common infusion solutions and containers were studied. During this study, the leaching of diethylhexyl phthalate (DEHP), a major plasticizer of some polyvinyl chloride (PVC) materials was also investigated. A formulation of topotecan hydrochloride was added to 50 ml PVC infusion bags, polyolefin infusion bags and 150 ml glass bottles containing either 5% dextrose injection or 0.9% sodium chloride injection at an initial nominal topotecan concentration of 0.05 mg ml −1. Additionally, the topotecan hydrochloride formulation was added to 50 ml PVC infusion bags containing either 5% dextrose injection or 0.9% sodium chloride injection at an initial nominal topotecan concentration of 0.025 mg ml −1. Containers were maintained at 5°C for 7 days or 23–24°C for 24 h. Samples were analyzed using a stability-indicating HPLC method to determine the concentration of topotecan and the presence of any degradates. The samples were also analyzed by separate HPLC methods to detect the presence of DEHP and the hydrolyzed lactone ring form (SKF 105992) of topotecan hydrochloride. In addition, the pH of each sample was measured initially and at the end of the storage time. There was no significant loss of topotecan observed for any of the conditions studied and no significant increase in degradates was observed. The pH remained unchanged for all samples between the start and end of the study. At the concentrations studied, topotecan hydrochloride was stable for up to 24 h at room temperature and for up to 7 days at 5°C, in PVC and polyolefin infusion bags and glass bottles containing either 5% dextrose injection or 0.9% sodium chloride injection. The presence of topotecan hydrochloride did not contribute to leaching of DEHP in the PVC infusion bags.

ChemInform Abstract: Rapid and Efficient Synthesis of High‐Purity Fluorine‐18 Labeled Haloperidol (V) and Spiperone (VII) via the Nitro Precursor in Combination with a New HPLC Separation Method.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Rapid and Efficient Synthesis of High-Purity Fluorine-18 Labeled Haloperidol and Spiperone via the Nitro Precursor in Combination with a New HPLC Separation Method

Abstract We have completed a convenient synthesis of fluorine-18 labeled butyrophenone neuroleptics from their nitro precursors. Thus, we have developed an efficient single-column HPLC system using a C18-bonded vinyl alcohol copolymer gel (octadecyl polymer, ODP) column and strongly alkaline solvent systems for purifying of the 18F-labeled butyrophenone neuroleptics obtained by a single-step 18F-for-nitro exchange reaction. The method has been applied to the synthesis of two typical butyrophenone neuroleptics ([18F]haloperidol and [18F]spiperone) with high purity in high yield. With information concerning the optimized conditions for the 18F-for-nitro exchange reaction, the synthetic method would be useful for synthesizing various 18F-labeled compounds.

High-performance flow atomic spectrometry: New nebulization techniques, on-line speciation and on-line sample pretreatment.

An interface-free combination of HPLC separation techniques and methods for element determination by atomic spectrometry can be achieved by hydraulic high-pressure nebulization (HHPN). With high-temperature HHPN (300 ( degrees )C) super heated liquids can be nebulized providing aerosol yields of up to 90% in flame AAS. This new nebulization method combines the advantages of HHPN and thermospray techniques (very small aerosol droplets, high aerosol yield, nebulization of saturated salt solutions).