Development and Validation of an SPE-HPLC-FL Method for the Determination of Anatoxin-a in Water and Trout (Oncorhincus mykiss)

Anatoxin-a is a neurotoxic alkaloid which is produced by various cyanobacteria genera and it is highly toxic to animals. Anatoxin-a risk evaluation is not fully determined yet. Therefore, it is imperative to implement detection and quantification methods in the various possible matrices. The recent worldwide shortage of acetonitrile, as well as environmental reasons, demand an urgent alternative to this high-performance liquid chromatography (HPLC) mobile phase solvent. This work describes an internal study that validated the measuring method of anatoxin-a in water and lyophilized trout matter by means of HPLC with fluorimetric detection (FLD) utilizing methanol as mobile phase.     

Quantitative and pharmacokinetic analysis of naloxone in plasma using high-performance liquid chromatography with electrochemical detection and solid-phase extraction

In this study we present a method for measuring naloxone in plasma after intravenous and oral administration of naloxone to humans, in order to study its pharmacokinetic profile. The method consists of a solid-phase extraction step followed by detection on a high-performance liquid chromatographic (HPLC) system equipped with an electrochemical dual-electrode detector. The extraction step employs cyanopropyl columns optimized for naloxone extraction to allow for elution of naloxone by the HPLC mobile phase; this eluate is then directly injected in the HPLC instrument. The HPLC system employs a radial compression phenyl column with a mobile phase containing 18% (v/v) acetonitrile and pentanesulfonic acid as ion-pairing agent; this system shows extraordinary high plate counts for naloxone. The detection limit is 3 ng (signal-to-noise ratio = 3) free naloxone per ml plasma. Following intravenous injection of 30 mg naloxone hydrochloride in two subjects, it was possible to determine the free naloxone concentration in the plasma for 8 h, more than four times the half-life of naloxone in plasma in humans.     

Simultaneous liquid chromatographic determination of seventeen of the major monoamine neurotransmitters, precursors and metabolites. I. Optimization of the mobile phase using factorial designs and a computer program to predict chromatograms

Utilizing reversed-phase high-performance liquid chromatography (HPLC) with electrochemical detection and optimization of the mobile phase using factorial designs and a constructed computer program to predict chromatograms, it has been possible to obtain a satisfactory resolution of seventeen of the major monoamine neurotransmitters, precursors and metabolites. A rapid (less than 25 min) isocratic system for the simultaneous determination of 3,4-dihydroxyphenylalanine, dopamine, dihydroxyphenylacetic acid, 3-methoxytyramine, homovanillic acid, norepinephrine, normetanephrine, 3,4-dihydroxyphenylethylene glycol, 3-methoxy-4-hydroxyphenylethylene glycol, epinephrine, metanephrine, vanillylmandelic acid, 5-hydroxytryptophan, serotonin, 5-hydroxytryptophol and 5-hydroxyindoleacetic acid in addition to the internal standard isoproterenol is presented. The optimization strategy included selection of variables to optimize by a reduced factorial design a detailed study of these variables by a complete factorial design, theoretical predictions of chromatograms by a constructed computer program and test on the HPLC system. This optimization strategy can easily be applied to any problem of solute separation by liquid chromatography.     

Determination of UV-transparent compounds by liquid chromatography using the UV detector

An indirect UV photometric detection technique is described in which a low concentration of a UV-absorbing compound (UVAC) is added to the mobile phase in reversed phase liquid chromatography, thereby making it possible for non UV-absorbing compounds such as the lower alcohols to be detected by the UV detector. This happens because the injected analyte may extract a portion of the UV absorbing compound from the mobile and/or stationary phase and the complex is co-eluted as a positive peak at the retention time of the analyte. Alternatively, the injected analyte may appear as a negative peak if the UV-absorbing compound is transferred to the mobile and stationary phases. In any case, the injected compound appears either as a positive or negative peak depending on the relative polarities and concentrations of all the compounds in the system. In addition, the resulting excess or deficiency of detection agent in the stationary phase is eluted separately as a positive or negative peak, indicating that the system has returned to equilibrium. In the work described herein, the chromatographic conditions and variables of the indirect photometric technique were studied to develop a quantitative HPLC method for UV-transparent compounds. It was found that under optimal conditions it is possible to determine some analytes quantitatively at concentrations as low as 0.05%.     

Retention behavior of polycyclic aromatic hydrocarbons in supercritical fluid chromatography on a chemically bonded stationary phases based upon liquid-crystalline polymer

Summary The retention behavior of a set of polycyclic aromatic hydrocarbons in supercritical fluid chromatography have been studied on a chemically bonded stationary phase based upon a side chain liquid crystalline polymer (LCP) with carbon dioxide-based mobile phase. The effects of the mobile phase pressure, column temperature and amount of mobile phase organic modifier have been investigated in order to detect a possible structural change in the liquid crystal polymer linked to the silica support. The influence of these factors on the selectivity coefficients has also been studied. Two distinctive behaviors with temperature are noted at low pressure on the one hand and at higher pressure on the other. This change in behavior is based on the density of the supercritical CO₂ and the PAH volatility rather than on any specific stationary phase structural change. Both lower mobile phase pressure and amount of mobile phase modifier are required to obtain better selectivities. Better planarity recognition is observed in SFC than in HPLC with these new bonded liquid crystal stationary phases. The bonded liquid crystal phase is only weakly affected by the addition of organic modifier in the supercritical CO₂.     

Separation mechanism exploration on metal-MBTAE-salicylic acid mixed ligand complexes by reversed-phase high performance liquid chromatography

The separation and determination of platinum metal and co-existing metal complexes by reversed-phase high performance liquid chromatography (HPLC) with 2-(6-methyl-2-benzothiazolylazo)-5-diethylamino phenol (MBTAE) as a precolumn derivatizing reagent is presented. The separation mechanism of these complexes was investigated by combining spectrophotometry with HPLC while salicylic acid was contained in the mobile phase. The results show that most platinum metal ions, Co(II) and Cu(II) can form ternary mixed ligand complexes with MBTAE and salicylic acid. The relationship of the retention behavior of complexes and the surface tension of the mobile phase (gamma), the column temperature (T), and the composition and space configuration of complexes was also investigated. Some possible configurations of complexes are also proposed. These may all be illustrated well from the viewpoint of solvophobic theory. These method allowed the prediction of the composition and structure of metal complexes by utilizing HPLC.     

Physicochemical properties of enhanced-fluidity liquid solvents

Enhanced fluidity (EF) liquid mixtures are advantageous as mobile phases for the separation of moderate to polar compounds in liquid chromatography (reversed-phase, normal, size exclusion, size exclusion, and chiral separations). The low viscosities and high diffusivities of EF mixtures allow highly efficient separations to be achieved in a small amount of time. The best use of enhanced-fluidity liquids is only possible when their physicochemical properties are known. Herein, the techniques used to measure the physicochemical properties (phase diagram, diffusivity, solvent strength and pH) of EF liquids are described. For each technique, the experiment design and the care necessary to insure the quality of the collected data are described. Finally, the impact of the measured physicochemical properties on the chromatography is also highlighted.     

Speciation of iron in breast milk and infant formulas whey by size exclusion chromatography-high performance liquid chromatography and electrothermal atomic absorption spectrometry

Speciation of iron in milk was carried out by high performance liquid chromatography (HPLC) and electrothermal atomic absorption spectrometry (ETAAS). Milk whey was obtained and low molecular weight protein separation was performed by size exclusion chromatography (SEC) with a TSK Gel SW glass guard (Waters) pre-column and a TSK-Gel G2000 glass (Toso Haas) column. After studying water as a possible mobile phase, this mobile phase was carefully selected in order to avoid alterations of the sample and to make subsequent iron determination in the protein fractions easier by ETAAS. The proposed method is sensitive (limit of detection [LOD] and LOQ 1.4 and 4.7 μg l⁻¹, respectively) and precise (relative standard deviation [RSD]<10%). Iron is principally found in the proteins of 3 and 76 kDa in breast milk, and it is irregularly distributed in infant formulas.     

Analysis of complex samples by solvating gas chromatography (supercritical fluid to gas transition)

The various forms of chromatography are primarily determined by differences in the physical state of the mobile phases. The main chromatographic categories include gas chromatography (GC), liquid chromatography, and supercritical fluid chromatography. Adjusting a temperature and pressure will change the mobile phase from liquid to supercritical fluid to gas, with concomitant changes in their physical properties. In this paper, the technique transition-phase chromatography (TPC) is described. In TPC, different mobile phase conditions exist inside the column. This phase transformation within the column results in huge differences in density, solvating power, viscosity, diffusivity, and, as a consequence, in the chromatographic properties of the mobile phase. TPC experiments using capillary columns packed in our laboratory have shown that when the mobile phase is transformed from supercritical fluid to gas, high column efficiencies can be achieved. The transition from supercritical fluid to gas (also called solvating GC), a particular case of the TPC, is evaluated for the separation of complex real samples (environmental, food, and fuels).     

Improvement of chemical analysis of antibiotics. X. Determination of eight tetracyclines using thin-layer and high-performance liquid chromatography

Analytical methods for eight tetracyclines (TCs) were established using silica gel high-performance thin-layer chromatography (HPTLC), reversed-phase thin-layer chromatography (RP-TLC) and high-performance liquid chromatography (HPLC). Good separations of eight TCs were obtained using chloroform-methanol-5% disodium ethylenediaminetetraacetate solution (65:20:5) (lower layer) and methanol acetonitrile 0.5 M oxalic acid solution (1:1:4) (pH 3.0) on silica gel HPTLC and C8 TLC plates, respectively. A combination of HPTLC and RP-TLC made possible the identification of the eight TCs. Each calibration graph was linear between 0.1 and 1.0 microgram using UV densitometry except for rolitetracycline. For detection reagents, the diazonium salts including Fast Violet B gave variously coloured spots with the eight TCs and good sensitivities were obtained except with minocycline. In HPLC, the simultaneous analysis of the eight TCs on a C8 column was possible using methanol-acetonitrile-0.01 M oxalic acid solution (1:1.5:7) adjusted to pH 3.0 as the mobile phase. A linear relationship was obtained between 1.0 and 10 ng using the usual sample preparation except for rolitetracycline. The direct determination of rolitetracycline was possible using tetrahydrofuran, dimethyl sulphoxide and the mobile phase as solvents for preparation of the sample. For the determination of residual rolitetracycline, it was effective to measure the amount of rolitetracycline as tetracycline by HPLC, HPTLC and RP-TLC after conversion of rolitetracycline to tetracycline by incubating for 5 min in methanol at 50 degrees C.     

Theoretical and experimental comparison of mobile phase consumption between ultra-high-performance liquid chromatography and high performance liquid chromatography

Ultra performance liquid chromatography (UPLC) using small particles and very high pressure has demonstrated higher resolution and speed compared with conventional HPLC. An additional benefit of UPLC is the significantly reduced consumption of mobile phase. This report discusses how column length, particle size, inner column diameter, extra column void volume, and capacity factor contribute to the reduction of mobile phase consumption in UPLC compared with HPLC. In addition, theoretical and experimental comparison of mobile phase consumption was made between isocratic HPLC and UPLC as well as between gradient HPLC and UPLC. Both theoretical and experimental results indicate that UPLC typically saves at least 80% of mobile phase in isocratic and gradient conditions when compared with HPLC.     

乙醇-水作为反相高效液相色谱流动相的研究

测定了乙醇在不同温度下的粘度,比较了乙醇与甲醇的理化性质和作反相高效液相色谱（ＲＰ ＨＰＬＣ）溶剂的特点。用乙醇 水作ＲＰ ＨＰＬＣ流动相测定中药有效成分,并将测定结果与甲醇 水或乙腈 水作流动相的测定结果进行比较。研究结果表明,选择合适的柱温等色谱条件,乙醇一般可以代替甲醇或乙腈用作ＲＰ ＨＰＬＣ流动相。     

Spherical coordinate representations of solvent composition for liquid chromatography method development using experimental design

One of the major techniques used for the method development of ternary and quaternary high performance liquid chromatography (HPLC) systems has been to use mixture designs, often referred to as "Glajch's Triangle". This technique does not allow for the systematic and simultaneous optimization of other factors such as gradient time, pH and temperature that affect the quality of separations. An alternative approach is to use experimental designs. The condition, however, that the composition of all components of the mobile phase must total 100% presents a problem when trying to mathematically represent ranges of each mobile phase constituent of a ternary or quaternary system. A method is described here, based on spherical coordinate representations, that adheres to the constraints of the mobile phase composition and allows experimental designs, such as central composite and factorial designs, to be applied to the simultaneous optimization of the mobile phase composition. Other factors, in particular temperature and gradient time, can then be included in the design. As a result of applying these designs to the HPLC separation of phenols and corticosteroids, it was found necessary to include three-way interactions between experimental factors in the model. The significance of these interactions shows that they need to be considered in HPLC method development.     

Artifactual-free analysis of S-nitrosoglutathione and S-nitroglutathione by neutral-pH, anion-pairing, high-performance liquid chromatography. Study on peroxynitrite-mediated S-nitration of glutathione to S-nitroglutathione under physiological conditions

The endogenous potent vasodilators and inhibitors of platelet aggregation S-nitrosoglutathione (GSNO) and S-nitroglutathione (GSNO2) are frequently analyzed by high-performance liquid chromatography (HPLC) using mobile phases of acidic pH. These systems are associated with problems stemming from rapid and considerable artifactual formation of GSNO from glutathione (GSH) and ubiquitous nitrite. We describe a novel ion-pairing HPLC method with UV absorbance detection at 334 nm for the highly specific and interference-free analysis of GSNO and GSNO2 in the presence of high GSH and nitrite concentrations. Complete avoidance of artifactual formation of GSNO was accomplished by using the anion-pairing agent tetrabutylammoniumhydrogen sulphate in the mobile phase that enables analysis of GSNO at neutral pH, at which GSH and nitrite do not react to form GSNO. This HPLC system was used to study formation of GSNO2 from GSH and peroxynitrite under physiological conditions. We found by this HPLC system that peroxynitrite (0-300 microM) reacts with GSH (0-5 mM) to form GSNO2 at a mean yield of 2%. Analysis of the same samples by a cation-pairing HPLC system with acidic mobile phase (pH 2.0) revealed, however, GSNO plus GSNO2 formation of the order of 20% due to on column reaction of GSH with peroxynitrite-derived nitrite to form GSNO. Ammonium sulfamate is frequently used to remove nitrite from thiol-containing solutions under acidic conditions. By means of the anion-pairing HPLC system it is demonstrated that nitrite removal by this method is incomplete even when ammonium sulfamate is used at high concentrations. These findings underscore the absolute requirement of neutral pH conditions for the analysis of GSNO. The novel anion-pairing HPLC method should be useful to provide reliable data on formation, reaction and metabolism of GSNO and GSNO2 in biological fluids using various detectors including mass spectrometers.     

Complementary mobile-phase optimisation for resolution enhancement in high-performance liquid chromatography

An optimisation methodology in high-performance liquid chromatography (HPLC) is presented for the selection of two or more mobile phases having an optimal complementary resolution. The complementary mobile phases (CMPs) are selected in such a way that each one resolves optimally only some compounds in the mixture, while the remainder, resolved by the other mobile phase(s), can overlap among them. The methodology is based on the computation of a peak purity measurement for each solute, using an asymmetrical peak model for peak simulation. Two global resolution criteria (product of elementary resolutions and worst elementary resolution) and two methods for solving the problem (a systematic examination of all possible solute arrangements, and the use of genetic algorithms to expedite the calculation time) were used to find the optimal CMPs. The CMP optimisation methodology was applied to the resolution of a mixture of 10 diuretics and beta-blockers, which could not be resolved using a single mobile phase; virtual baseline resolution was achieved, however, with two CMPs.     

Enhanced‐fluidity liquid chromatography using mixed‐mode hydrophilic interaction liquid chromatography/strong cation‐exchange retention mechanisms

The potential of enhanced‐fluidity liquid chromatography, a subcritical chromatography technique, in mixed‐mode hydrophilic interaction/strong cation‐exchange separations is explored, using amino acids as analytes. The enhanced‐fluidity liquid mobile phases were prepared by adding liquefied CO₂ to methanol/water mixtures, which increases the diffusivity and decreases the viscosity of the mixture. The addition of CO₂ to methanol/water mixtures resulted in increased retention of the more polar amino acids. The “optimized” chromatographic performance (achieving baseline resolution of all amino acids in the shortest amount of time) of these methanol/water/CO₂ mixtures was compared to traditional acetonitrile/water and methanol/water liquid chromatography mobile phases. Methanol/water/CO₂ mixtures offered higher efficiencies and resolution of the ten amino acids relative to the methanol/water mobile phase, and decreased the required isocratic separation time by a factor of two relative to the acetonitrile/water mobile phase. Large differences in selectivity were also observed between the enhanced‐fluidity and traditional liquid mobile phases. A retention mechanism study was completed, that revealed the enhanced‐fluidity mobile phase separation was governed by a mixed‐mode retention mechanism of hydrophilic interaction/strong cation‐exchange. On the other hand, separations with acetonitrile/water and methanol/water mobile phases were strongly governed by only one retention mechanism, either hydrophilic interaction or strong cation exchange, respectively.     

β-Diketones: Peak shape challenges and the use of mixed-mode high-performance liquid chromatography methodology to obtain fast,high resolution chromatography

Historically, indirect methods have been used for the HPLC analysis of β-diketone compounds because of the very poor peak shapes and resolution obtained on conventional HPLC stationary phases. In this paper we demonstrate that it is possible to obtain good peak shapes for underivatised β-diketone compounds, in a simulated reaction mixture, using only conventional mobile phases with mixed-mode stationary phase HPLC columns. Optimum conditions were obtained using the mixed-mode reversed-phase strong anion exchange column Primesep B, supplied by SIELC Technologies, with a 0.1% aq. TFA/MeOH gradient method and a column temperature of 55 °C.     

Quantitative determination of phospholipids in amniotic fluid by high-performance liquid chromatography

Summary A new quantitative analytical method for the determination of phospholipids in amniotic fluid by high performance liquid chromatography (HPLC) is described. In addition to the main compounds, phosphatidylcholine (lecithin) and sphingomyelin, the so-called minor phospholipids, phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine can also be determined. Separation is achieved using a guard-column of Lichrosorb Si 60 and an analytical column of Lichrosorb DIOL. Acetonitrile/water is used as mobile phase at an elevated temperature. By determining the recovery rates, the within-run and the between-run precision, it was shown that sufficient accuracy and precision could be achieved for all the parameters examined. The method is highly sensitive, the detection limit for sphingomyelin is 0.2 μg and 0.1 μg for all the other components. A single determination of 5 phospholipids in an amniotic fluid sample takes about two hours. By performing simultaneous extractions it is possible to analyse 5 samples per day.     

Rapid Separation of Proteins with Monodisperse Porous Polystyrene-Divinylbenzene(PS/DVB) Chromatographic Packings

It has been recognized for half a century that stagnant mobile phase mass transfer is a dominant limitation in liquid chromatography. The resolution is seriously affected at high mobile phase velocity. One approach to solve this problem is to eliminate the pores. Through the use of 1~2 μm non-porous particles it has been possible to carry out protein separations an order of magnitude faster,albeit at the expense of diminished loading capacity and high operating pressure. A second alternative is to cause liquid to flow through the particles. Because convective transports is much more rapid than that achieved by diffusion, these materials can also be used at an order of magnitude higher velocity for mobile phase in protein separations.     

High-performance liquid and gas-liquid chromatography of isomeric phenoxathiins. Use of organo-clay to increase selectivity

Summary Bentone-34, a selective adsorbent, has been studied for use in high-performance liquid chromatography (HPLC) to separate isomeric phenoxathiin derivatives. We have compared the performance of this adsorbent in gas chromatography and liquid chromatography. We have obtained better and faster analyses of these isomeric phenoxathiins by high-pressure liquid-solid chromatography. This method is more flexible, as we have the choice of a large variety of mobile phases, and analyses are achieved at ambient temperature. Moreover HPLC can be used for collecting the pure solutes.