Application of High Temperature LC to the Separation of AZD5438 (4-(1-Isopropyl-2-methyl-1H-imidazol-5-yl)-N-[4-(methylsulfonyl)phenyl]pyrimidin-2-amine) and Its Metabolites: Comparison of LC, UPLC and HTLC

A comparison is made of the separation and analysis of a test probe, AZD5438 (4-(1-isopropyl-2-methyl-1H-imidazol-5-yl)-N-[4-(methylsulfonyl)phenyl]pyrimidin-2-amine), and its metabolites using conventional LC, UPLC and high temperature liquid chromatography (HTLC). LC and UPLC separations were performed using reversed-phase water:acetonitrile solvent systems whilst HTLC was performed with an entirely aqueous mobile phase, using both isothermal and temperature gradient chromatography. Substantial reductions in run times were observed with both UPLC and HTLC compared to the conventional LC approach without loss in chromatographic resolution for the major metabolites. At temperatures in excess of 100 °C, thermal degradation of some of the metabolites was observed in an isothermal separation, however, the application of a thermal gradient proved successful in maintaining the structural integrity of the analytes and simultaneously separating the parent compound and its major metabolites.     

Practice and theory of high temperature liquid chromatography

High temperature liquid chromatography (HTLC) exists in a temperature region beyond ambient (ca. 40 degrees C) and below super critical temperatures. The promises of HTLC, such as increased analysis speed, enhanced separation productivity, "green" LC with pure water mobile phases coupled to universal FID detection, and fast analysis of complex samples by combination with fast 2-D techniques, have become an option for routine practice. The focus of this paper is to review the key developments that have made the application of HTLC a practical technique and draw attention to new developments in 2-D techniques that incorporate HTLC that offer an opportunity to vastly increase the usefulness of HPLC for the analysis of complex samples.     

Quantification of the In Vitro and In Vivo Metabolic Fates of 2-, 3- and 4-Bromobenzoic Acids Using High Temperature LC Coupled to ICP-MS and Linear Ion Trap MS

High temperature liquid chromatography (HTLC), with water as the mobile phase, combined with ICP-MS tuned to the detection of Br, for quantification, and a linear ion trap MS, for structural identification, were applied to determine the disposition and metabolic fate of 2-, 3- and 4-bromobenzoic acids (BBAs) following in vitro incubation with rat hepatocytes at 4 mM. The separation of the metabolites was performed using a thermal gradient to increase the eluotropic strength of the aqueous mobile phase through the run to elute less polar components. The use of highly aqueous solvents for separations involving ICP-MS is advantageous because the water does not change the conductivity of the plasma thereby providing a more stable system. The improved system stability resulted in better sensitivity, as shown by the increased signal intensity for HTLC compared to conventional reversed-phase separations. Using HTLC to investigate the in vitro metabolic fate of the BBAs showed the major route of metabolism to be glycine conjugation, irrespective of the structure of the parent, but with different amounts produced depending on the positional isomer. The comparison of HTLC with the conventional methodology showed that chromatography at elevated temperatures had no effect on the observed metabolite profile. HTLC was also applied to urine obtained from an in vivo sample and showed an improved chromatographic peak shape compared to conventional liquid chromatography (LC) whilst providing the same analytical result.     

High-Throughput Determination of Quinine in Beverages and Soft Drinks Based on Zone-Fluidics Coupled to Monolithic Liquid Chromatography

In the present study we propose a new automated analytical scheme for the determination of quinine in soft drinks and beverages by coupling zone-fluidics (ZF) to liquid chromatography (LC). Certain pretreatment tasks involving precise liquid handling, such as sample dilution and addition of the internal standard (ISTD) were automated by ZF. After on-line filling of the loop of the LC injection valve, separation was carried out in less than 2.5 min by using a reversed phase monolithic column (100 × 4.6 mm i.d.) at a flow rate of 2 mL min^?1. The ZF and LC parts of the setup operated independently: during LC separation the next sample was treated by ZF allowing a high throughput of 24 h^?1. The proposed assay was thoroughly validated in terms of linearity (up to 150 mg L^?1), LOD (0.25 mg L^?1), LOQ (0.8 mg L^?1), precision, accuracy, and ruggedness. The ZF-LC method was successfully applied for the determination of quinine in beverages and soft-drinks.     

Regulatory Aspects in Development of Stability-Indicating Methods: A Review

Poly(N-vinylcaprolactam) (PVCL) connected to aminopropyl silica is a new stationary phase for temperature responsive liquid chromatography (TR-LC). PVCL shows a transition from hydrophilic to hydrophobic interaction between 30 and 40 °C. The synthesis is described in detail. The temperature responsive characteristic of the phase is illustrated with a mixture of steroids using pure water as mobile phase. An increase in retention is observed when raising the temperature. H–u plots at different temperatures were constructed. Below the lower critical solution temperature (LCST), no optimal velocity could be measured because of substantial resistance to mass transfer. Above the LCST, u _opt was ca. 0.3 mm s^?1 with reduced plate heights from 4 at 45 °C to 3 at 65 °C. The temperature responsive nature of the polymer is lost in green chromatography with ethanol as modifier in concentrations above 5%.     

UPLC a Powerful Tool for the Separation of Imidazolium Ionic Liquid Cations

Ultra-performance liquid chromatography (UPLC) in reversed-phase (RP), ion pair (IP) and hydrophilic interaction chromatography (HILIC) has been investigated for the separation of imidazolium-based ionic liquid (IL) cations. Among the three stationary phases (i.e., C18, C8 and phenyl) studied under RP conditions the phenyl phase provided much stronger retention for the IL cations. Four acids (hydrochloric, methanesulfonic, perchloric and trifluoroacetic) as mobile phase additives were compared in light of their effects on the retention of IL cations. It was shown that the retention of all IL cations decreased upon acidification of the mobile phase, possibly due to suppression of residual silanol ionization. Very fast (~3 min) and efficient RP-UPLC separation of six cations was achieved by gradient elution with acetonitrile?Cwater mobile phase containing 2.5 mmol L^?1 perchloric acid. In IP-UPLC all solutes were well resolved in about 4 min by gradient elution with acetonitrile?Cwater mobile phase containing 1 mmol L^?1 sodium 1-octanesulfonate as ion pairing reagent. Finally, under HILIC conditions by using isocratic elution with acetonitrile?Cwater (85:15, v/v) mobile phase containing 5 mmol L^?1 ammonium formate (pH 3.2) the separation time was reduced to less than 2 min while maintaining excellent peak shapes and sufficient resolution. Compared to current LC systems UPLC allowed considerably faster separations with better peak shapes.     

HPLC–DAD–FLD Determination of Veterinary Pharmaceuticals in Pharmaceutical Industry Wastewater with Precolumn Derivatization Using Fluorescamine

A simple and robust method suitable for routine analysis of pharmaceuticals from different classes in pharmaceutical industry wastewater samples is presented. Seven veterinary pharmaceuticals (antibiotics and anesthetic) were simultaneously isolated from a highly complex wastewater matrix. Target compounds were three sulfonamide antibiotics (sulfaguanidine, sulfadiazine, and sulfamethazine), three fluoroquinolone antibiotics (ciprofloxacin, enrofloxacin, norfloxacin), and one anesthetic, procaine. The investigated compounds were simultaneously pre-concentrated and cleaned up by solid-phase extraction using Strata-X extraction cartridges. The analysis was performed using liquid chromatography (LC) with diode array and fluorescence detectors connected in series to the chromatographic system. LC separation was performed on a C18 modified column (Phenomenex) with a gradient elution of mobile phase (0.1 % acetic acid in water and 0.1 % acetic acid in acetonitrile) in 25 min at 30 °C. Recoveries ranged from 86.9 to 110 % with relative standard deviations below 10.1 %. Method limits of quantification were in the range of 0.005–0.1 μg L^?1 depending on the fluorescence intensity after precolumn derivatization by fluorescamine.     

LC Analysis of Aliphatic Primary Amines and Diamines After Derivatization with 2,6-Dimethyl-4-quinolinecarboxylic Acid N-Hydroxysuccinimide Ester

A method for sensitive simultaneous analysis of aliphatic primary amines and diamines has been developed and validated. The compounds were analyzed by reversed-phase high-performance liquid chromatography after pre-column derivatization with 2,6-dimethyl-4-quinolinecarboxylic acid N-hydroxysuccinimide ester as fluorescent probe. The derivatization reaction was performed at 50 °C for 40 min in 0.1 mol L^?1 borax buffer solution (pH 7.5). The resulting fluorophores were separated to baseline on a C₁₈ column and fluorimetrically detected at λ _ex/λ _em = 326/409 nm. Detection limits were in the range 0.50–0.02 nmol L^?1. The method was successfully used for analysis of aliphatic amines in water, human urine, and serum.     

A Simple Method for Determination of Homologue Imidazolium Cations in Ionic Liquids Using IC with Direct Conductivity Detection

An investigation was carried out into the fast determination of five homologue imidazolium cations in ionic liquids by ion chromatography using a cation-exchange column and direct conductivity detection. Ethylenediamine, complex organic acid (citric acid, oxalic acid and tartaric acid) and organic modifiers (acetonitrile) were used as mobile phase. The influences of the eluent types, eluent concentration, eluent pH and column temperature on separation of the cations were discussed. Simultaneous separation and determination of the five homologue imidazolium cations in ionic liquids were achieved under an optimum condition. The optimized mobile phase was consisted of 0.25 mmol L^?1 ethylenediamine + 0.5 mmol L^?1 citric acid + 3% acetonitrile (v/v) (pH 4.1), set at a flow rate of 1.0 mL min^?1. The column temperature was 40 °C and detection limits were obtained in the range of 1.1–45.6 mg L^?1. The relative standard deviations of the chromatographic peak areas for the cations were <3.0% (n = 5). This method was successfully applied to separate imidazolium cations in ionic liquids produced by organic synthesis. The recoveries of spiked components were 92.5–101.9%.     

High-temperature reversed-phase liquid chromatography coupled to isotope ratio mass spectrometry

Compound‐specific isotope analysis (CSIA) by liquid chromatography coupled to isotope ratio mass spectrometry (LC/IRMS) has until now been based on ion‐exchange separation. In this work, high‐temperature reversed‐phase liquid chromatography was coupled to, and for the first time carefully evaluated for, isotope ratio mass spectrometry (HT‐LC/IRMS) with four different stationary phases. Under isothermal and temperature gradient conditions, the column bleed of XBridge C₁₈ (up to 180 °C), Acquity C₁₈ (up to 200 °C), Triart C₁₈ (up to 150 °C), and Zirchrom PBD (up to 150 °C) had no influence on the precision and accuracy of δ¹³C measurements, demonstrating the suitability of these columns for HT‐LC/IRMS analysis. Increasing the temperature during the LC/IRMS analysis of caffeine on two C₁₈ columns was observed to result in shortened analysis time. The detection limit of HT‐RPLC/IRMS obtained for caffeine was 30 mg L^–1 (corresponding to 12.4 nmol carbon on‐column). Temperature‐programmed LC/IRMS (i) accomplished complete separation of a mixture of caffeine derivatives and a mixture of phenols and (ii) did not affect the precision and accuracy of δ¹³C measurements compared with flow injection analysis without a column. With temperature‐programmed LC/IRMS, some compounds that coelute at room temperature could be baseline resolved and analyzed for their individual δ¹³C values, leading to an important extension of the application range of CSIA. Copyright © 2011 John Wiley & Sons, Ltd.     

Applying thermodynamics to digestion/gasification processes: the Attainable Region approach

The research shows theoretical calculations on the thermodynamics of digestion/gasification processes where glucose is used as a surrogate for biomass. The change in Enthalpy (?H) and Gibbs Free Energy (?G) is used to obtain the Attainable Region (AR) that shows the overall thermodynamic limits for digestion/gasification from 1 mol of glucose. Gibbs Free Energy and Enthalpy (G–H) plots were calculated for the temperature range 25–1500 °C. The results show the effect of temperature on the AR for the processes when water is in both liquid and gas states using 25 °C, 1 bar as the reference state. The AR results show that the production of CO, H₂, CH₄ and CO₂ are feasible at all temperatures studied. The minimum Gibbs Free Energy becomes more negative from ?418.68 kJ mol^?1 at 25 °C to ?3024.34 kJ mol^?1 at 1500 °C while the process shifts from exothermic (?141.90 kJ mol^?1) to endothermic (1161.80 kJ mol^?1) for the respective temperatures. Methane and carbon dioxide are favoured products (minimum Gibbs Free Energy) for temperatures up to about 600 °C, and this therefore includes Anaerobic Digestion. The process is exothermic below 500 °C, and thus Anaerobic Digestion requires heat removal. As the temperature continues to increase, hydrogen production becomes more favourable than methane production. The production of gas is endothermic above 500 °C, and it needs a supply of heat that could be done, either by combustion or by electricity (plasma gasification). The calculations show that glucose conversion at temperatures around 700 °C favours the production of carbon dioxide and hydrogen at minimum G. Generally, the results show that the gas from high-temperature gasification (>~800 °C) typically carries the energy mainly in syngas components CO and H₂, whereas at low-temperature gasification (<500 °C) the energy is carried in CH₄. The overall analysis for the temperature range (25–1500 °C) also suggests a close relationship between biogas production/digestion and gasification as biogas production can be referred to as a form of low-temperature gasification.     

Development of selective comprehensive two-dimensional liquid chromatography with parallel first-dimension sampling and second-dimension separation—application to the quantitative analysis of furanocoumarins in apiaceous vegetables

Various implementations of two-dimensional high-performance liquid chromatography are increasingly being developed and applied to the analysis of complex materials, including those encountered in the analysis of foods, beverages, and nutraceuticals. Previously, we introduced the concept of selective comprehensive two-dimensional liquid chromatography (sLC?×?LC) as a hybrid between the more conventional, but extreme opposite sampling modes of heartcutting (LC–LC) and fully comprehensive (LC?×?LC) 2D separation. The sLC?×?LC approach breaks the link between first dimension (¹D) sampling time and second dimension (²D) analysis time that is faced in LC?×?LC and allows very rapid (as low as 1 s) sampling of highly efficient ¹D separations, while at the same time allowing efficient ²D separations on the timescale of tens of seconds. In this paper, we improve upon our previous sLC?×?LC work by demonstrating the ability to perform the processes of ¹D sampling and ²D separation in parallel. This significantly improves the flexibility of the technique and allows targeted analysis of analytes that elute close together in time in the ¹D separation. To demonstrate the value of this added capability, we have developed a sLC?×?LC method using multi-wavelength ultraviolet absorbance detection for the quantitative analysis of six target furanocoumarin compounds in extracts of celery, parsley, and parsnips. We show that ²D separations of ¹D effluent containing the target compounds of interest reveal the presence of unanticipated interferent peaks that would otherwise compromise the quantitative accuracy of the method. We also demonstrate the application of the chemometric method iterative key set factor analysis with alternating least-squares to sLC?×?LC to mathematically resolve target compounds that are only slightly separated chromatographically but not sufficiently resolved for accurate quantitation.     

Rapid LC-UV Analysis of Iohexol in Canine Plasma for Glomerular Filtration Rate Determination

A rapid high-performance liquid chromatography UV method and a simple sample preparation for analyzing iohexol in canine plasma, for evaluating glomerular filtration rate (GFR) and intestinal permeability, were developed and validated. Trifluoroacetic acid (TFA) was used for protein precipitation and iohexol extraction from plasma, followed by vortex mixing and centrifugation. As an internal standard, 4-aminobenzoic acid (para-aminobenzoic acid, PABA) was added. The supernatant (5 μL) was injected into a Zorbax SB-C18 LC column maintained at 50 °C. The mobile phase of the LC method was a water–methanol gradient at pH 3.0 adjusted with TFA. Fast LC measurement was achieved by using a rapid-resolution LC technique. Total run time was 13 min, and UV wavelength was set at 246 nm. Precision of the method was 0.2–9.0%, depending on the iohexol concentration in plasma. Recovery of iohexol from plasma was over 90%, and recovery of the internal standard 99.1 ± 1.4%. The calibration curve was linear (r = 0.9997) over iohexol concentrations of 2.5–150 μg mL^?1 (n = 5). This method is fast, simple, reliable and applicable in clinical settings.     

High-Temperature Liquid Chromatography

Abstract

The influence of temperature on separation is discussed. Elevated temperature can significantly reduce the retention times in liquid chromatography and thus the throughput and running costs of a LC instrument. A decrease of viscosity at increasing temperatures allows the use of columns packed with very small particles and of higher flow rates as well. Modern reversed phase columns are stable up to (and >) 90°C. Metal oxide columns (Zirconium oxide, Titanium dioxide, Aluminium oxide) and polymer packed columns are temperature stable up to 200°C. The consumption of organic solvents can significantly be reduced and thus also the disposal costs.     

Biphenyl-based liquid crystals for elevated temperature processing with polymers

Due to the limited thermal stability of current commercially available liquid crystals (LCs), the incorporation into polymer composites through standard processing techniques, such as melt coextrusion, has been hindered. Motivated by this dilemma, a series of smectic B liquid crystalline structures based on the 4,4?-alkyl substituted biphenyl moiety were synthesised through conventional methodologies and probed for their thermal stability and LC properties. Degradation temperatures were found to increase with increasing aliphatic chain length – up to 295 °C for C16 substituted structures, which is well above the processing temperatures of commercial polymers. Additionally, all compounds were found to be liquid crystalline in nature with crystal-to-smectic B transition temperatures ranging from 49.8 °C to 91.4 °C. Thermal stability, phase separation, and compatibility of LC/polystyrene composites were also examined. Less than 10% of 15A15 LC by weight in polystyrene exhibited good polymer miscibility, while phase separation occurred at loads higher than 15% by weight. We foresee the use of these LCs in applications that require elevated processing conditions to produce materials with enhanced mechanical or gas barrier properties.     

Characteristics and kinetics analysis of Codonopsis pilosula pyrolysis

Eight kinds of Radix Codonopsis (RC) from different origins in China were selected as the experimental samples fort his study. Their pyrolysis processes were researched by the method of thermogravimetry analysis, in which the heating course was set in the ways of programming temperature from room temperature to 500 °C at different heating rates. Research results show that the process in the heating period of RC includes three stages: water loss, fast pyrolysis, and medium rate decomposition. For cultivated RC, the average initial decomposition temperature in the fast pyrolysis stage is 115 °C, whereas the peak temperature of the fast pyrolysis stage is changed from 189 to 225 °C, in which stage the alcohol-soluble substances are mainly decomposed. It is required to control the operational temperatures of drying and concocting processes according to initial decomposition temperature. Kissinger–Akahira–Sunose model can be used to describe the process mechanism of RC pyrolysis, and the kinetic analyses based on the fast pyrolysis stage thermogravimetric data show that the activation energies change from 141 to 207 kJ mol^?1 for cultivated RC samples and 122 to 131 kJ mol^?1for wild RC samples. The alcohol-soluble extract (ASE) content of wild RC samples is lower than that of cultivated RC samples; their thermal stability is also relatively poor.     

Comparison of LC and UPLC Coupled to MS–MS for the Determination of Sulfonamides in Egg and Honey

Determination of ten sulfonamides (SAs) in egg and honey has been compared using column liquid chromatography (LC) and ultra-performance liquid chromatography (UPLC) coupled to tandem mass spectrometry (MS–MS). A liquid–liquid extraction with acetonitrile followed by solid-phase extraction on a Strata-X cartridge was developed for sample preparation. The analytical performance of both methods was compared applying the alternative matrix-comprehensive in-house validation approach using specially designed software InterVal?. Using UPLC the separation time was shortened about 30% reducing the run time by 8 min and a better resolution was achieved compared to LC. Due to higher peak efficiency achieved with UPLC, the decision limit values obtained by both techniques were almost equal (6.61–9.43 μg kg^?1 and 7.25–11.9 μg kg^?1 for UPLC and LC, respectively), despite the fact that in UPLC twice lower sample volumes were injected. Satisfactory and comparable recoveries (80–110%) were obtained by UPLC and LC for all the SAs, except for sulfacetamide by LC and sulfabenzamide by both methods. For a majority of the spiked compounds, UPLC gave significantly better precision.     

LC Method for the Determination of Rhaponticin in Rat Plasma, Faeces and Urine for Application to Pharmacokinetic Studies

A simple liquid chromatography (LC) method has been developed and validated to determine rhaponticin in rat plasma, faeces and urine. Chromatographic separation was achieved through mobile phase consisting of acetonitrile and water at a flow rate of 1.0 mL min^?1. Rhaponticin was quantified using UV detection at 324 nm. The assay was linear over the concentration range of 50–4,000 ng mL^?1 for plasma, faeces and urine. The intra- and inter-day RSD were less than 10%. The plasma, faeces and urine rhaponticin levels were monitored in rats after oral administration. This simple LC method appears to be useful in the pharmacokinetic investigation of rhaponticin.     

Sense and nonsense of high-temperature liquid chromatography

High-temperature liquid chromatography (HTLC) is recognized today as a valuable technique in reversed-phase high-performance liquid chromatography (RP-HPLC). Column temperature can play a role in reducing analysis time, modifying retention, controlling selectivity, changing efficiency or improving detection sensitivity. The different effects of high temperatures on reversed-phase separations, the practical limitations due to the instrumentation, the limits and the main advantages of HTLC, especially for the separation of polar and ionized compounds, are reviewed.     

Pyrolysis of Eucalyptus wood in a fluidized-bed reactor

Eucalyptus wood can be utilized as a biomass feedstock for conversion to bio-oil using a pyrolysis process. Eucalyptus wood samples were initially pyrolyzed on a laboratory-scale pyrolysis system at different values in the ranges of 300–800 °C and 0.050–0.300 L min^?1 to determine the effects of operation temperature and N₂ flow rate, respectively, on the yields of products. Then, the bio-oil in the highest yield (wB = 44.37 %), which was obtained at pyrolysis final temperature (450 °C), heating rate (35 °C min^?1), particle size (850 μm), and sweeping flow rate (0.200 L min^?1), was characterized by Fourier transform infra-red spectroscopy, gas chromatography/mass spectrometry and column chromatography. Subsequently, it was shown that the operating temperature and N₂ gas flow rate parameters affected the product yields. Also, some important physico-chemical properties of the pyrolytic oil obtained in high yield were determined as a calorific value of 37.85 MJ kg^?1, an empirical formula of CH_1.651O_0.105N_0.042S_0.001, a rich chemical content containing many different chemical groups, a density of 981.48 kg m^?3, and a viscosity of 61.24 mm² s^?1. Based on the determined properties of the pyrolytic oil, it was concluded that the use of pyrolytic oil derived from Eucalyptus wood may be useful for the production of alternative liquid fuels and fine chemicals after the necessary improvements.