Evaporative Light-scattering Detection System Research Articles

Determination of stearic acid and 12-hydroxyoctadecanoic acid in PEG-60 hydrogenated castor oil by HPLC-ELSD after alkaline hydrolysis

AbstractA rapid and simple method for the determination of stearic acid and 12-hydroxystearic acid in PEG-60 hydrogenated castor oil by high performance liquid chromatography with evaporative light scattering detection was established. The oil sample was first pretreated by alkaline hydrolysis. The analysis was performed on a Zhongpu Develop XD-C18 column (250 mm × 4.6 mm, 5 µm) with gradient elution of methanol and 1% acetic acid aqueous solution at a flow rate of 1.2 mL·min−1 and a column temperature of 40 °C. The drift tube temperature of the evaporative light scattering detection system was set at 40 °C, and the pressure of carrier gas (N2) was 337 kPa. The regression equation revealed a good linear relation (r = 0.9993–0.9995) during the test ranges (119.1–1190.7 μg·mL−1 for 12-hydroxystearic acid, 10.7–107.4 μg·mL−1 for stearic acid). The detection limits of 12-hydroxystearic acid and stearic acid were 1.1 and 2.5 μg·mL−1, the limits of quantitation were 3.2 and 7.4 μg·mL−1, respectively. And the mean recoveries were 101.5 and 101.0%, the corresponding relative standard deviations (RSDs) were 2.1 and 2.8%, respectively. The RSDs corresponding to repeatability (n = 6) were both less than 1.7% in terms of precision. As to the stability, the test results remained stable after 8 h at room temperature (RSDs were both less than 2.6%). The developed method showed high sensitivity, recovery, repeatability and stability, which indicated that the method could be applied as a quality evaluation method for the determination of stearic acid and 12-hydroxyoctadecanoic acid in PEG-60 hydrogenated castor oil.

Simultaneous Quantification of DPPG, DEPC and Cholesterol in Propofol Liposome by HPLC-ELSD Using Alkaline Hydrolysis.

A high-performance liquid chromatography method with evaporative light-scattering detection (ELSD) was performed for simultaneous determination of dipalmitoyl phosphatidylglycerol (DPPG), dierucoyl phosphatidylcholine (DEPC) and cholesterol in propofol liposome by the pretreatment of alkaline hydrolysis (temperature, concentration of KOH anhydrous ethanol solution and reaction time were 90°C, 1mol · L-1 and 10min, respectively). The analysis was carried out on an Agilent TC-C18 column (4.6mm×250mm, 5μm) with isocratic elution of methanol and 0.1% acetic acid aqueous solution (95:5, v/v) at a flow rate of 1.0mL· min-1. The column temperature was 30°C. The drift tube temperature of the ELSD system was set at 30°C, and the pressure of carrier gas was 350KPa. The regression equation revealed a good linear relationship (r=0.9990-0.9993) during the test ranges. The RSD of stability and repeatability (n=6) was found to be less than 1.96 and 1.46%, respectively. The average recoveries ranged from 97.90 to 101.00%. The proposed method was validated and showed good precision, stability, repeatability and recovery, which indicated that the method could be readily utilized as a quality evaluation method for the determination of DPPG, DEPC and cholesterol in propofol liposome.

Simultaneous separation and determination of fructose, sorbitol, glucose and sucrose in fruits by HPLC–ELSD

A high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was optimised for simultaneous determination of fructose, sorbitol, glucose and sucrose in fruits. The analysis was carried out on a Phenomenex Luna 5u NH2 100A column (250mm×4.60mm, 5micron) with isocratic elution of acetonitrile:water (82.5:17.5, v/v). Drift tube temperature of the ELSD system was set to 82°C and nitrogen flow rate was 2.0Lmin−1. The regression equation revealed good linear relationship (R=0.9967–0.9989) within test ranges. The limits of detection (LOD) and quantification (LOQ) for four analytes (peach, apple, watermelon, and cherry fruits) were in the range of 0.07–0.27 and 0.22–0.91mgL−1, respectively. The proposed HPLC–ELSD method was validated for quantification of sugars in peach, apple, watermelon, and cherry fruits, and the results were satisfactory. The results showed that the contents of the four sugars varied among fruits. While fructose (5.79–104.01mgg−1) and glucose (9.25–99.62mgg−1) emerged as common sugars in the four fruits, sorbitol (8.70–19.13mgg−1) were only found in peach, apple and cherry fruits, and sucrose (15.82–106.39mgg−1) were in peach, apple and watermelon. There was not detectable sorbitol in watermelon and sucrose in cherry fruits, respectively.

Quality control for Coptidis rhizoma through the determination of five alkaloids by HPLC–ELSD coupled with chemometrics

A simple, sensitive and reliable high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) has been developed for simultaneous determination of five major alkaloids in Coptidis rhizoma. Simultaneous separation of five alkaloids was achieved on a Kromasil C18 analytical column (250 mm × 4.6 mm, 5 µm) with the isocratic elution of acetonitrile : water (30 : 70, v/v, the pH was adjusted to 6.0 with 0.2 moL L−1 trichloroacetic acid). The drift tube temperature of the ELSD system was set at 115°C and the nitrogen flow rate was 2.8 L min−1. All the five calibration curves showed a good linear relationship (r 2 > 0.9991) within the test ranges. The limit of detections and quantifications for the five alkaloids in ELSD were less than 0.88 and 2.73 ng, respectively. The established method was successfully applied to quantify the five ingredients in different C. rhizoma samples and evaluate the internal quality of C. rhizoma samples from various sources by analysing the chemical fingerprints using similarity analysis (SA), hierarchical clustering analysis (HCA) and principal component analysis (PCA), which provided a useful basis of overall evaluation of the quality of C. rhizoma.

Analysis of Molecular Marker Compounds from <i>Vitexagnus cactus</i> Using the High Performance Liquid Chromatography and Evaporative Light Scattering Detector Techniques

Dried whole plant of Vitexagnus cactus was extracted with organic solvents in different ratios and analyzed using high performance liquid chromatography (HPLC) with evaporative light scattering (ELS) detector. The markers of interest in Vitexagnus castus were agnuside, casticin and vitexyl acetone. These standard molecular markers do not contain strong chromophores, hence it is difficult to identify them using the UV detection system in high performance liquid chromatography. ELS detector is regarded as a valuable alternative to UV detection system for identification of the compounds that do not contain strong chromophores. The results of the present study show that the ELS detection system is more efficient than the UV-visible detection system.Key words: Vitexagnus castus, evaporative light scattering detector, high performance

Simultaneous determination of sugars and picrosides in Picrorhiza species using ultrasonic extraction and high-performance liquid chromatography with evaporative light scattering detection

Sugars play a critical role in regulating overall cellular metabolism in high altitude growing plants. These plants are shown to have high levels of sugars to enhance their tolerance to abiotic stresses such as drought and freezing temperature. In the present study, a simple, sensitive, selective and reliable HPLC method based on ultrasonic extraction and evaporative light scattering detection (ELSD) has been developed and validated for the simultaneous determination of important sugars (xylose, xylitol, mannitol, glucose and sucrose) and picrosides (picroside-I and picroside-II) in two species Picrorhiza kurroa and P. scrophulariiflora. The analysis was carried out on a Zorbax amino column (250 mm × 4.6 mm i.d., 5 μm) with isocratic elution of acetonitrile:water (78:22, v/v). The method was validated for accuracy, precision, limit of detection and quantification according to International Conference on Harmonization (ICH) guidelines. The drift tube temperature of the ELSD system was set to 81 °C and nitrogen flow rate was 2.0 standard liter per minute (SLM). The regression equation revealed a good linear relationship ( r 2 = 0.9997 ± 0.0012) within test ranges. The limit of detection and quantification for seven analytes in ELSD were less than 0.98 and 2.95 μg, respectively. The method showed good reproducibility for the quantification of seven analytes in Picrorhiza species with intra- and inter-day variation of less than 2.0%.

Quantitative evaluation of sebum lipid components with nuclear magnetic resonance

A NMR spectroscopic method is described that enables the quantitation of specific lipid classes and components, independent of fatty acid composition. We demonstrate this method for measuring cholesterol, squalene, and pools of sterol esters, wax esters (WEs), and triglyceride (TG) components in sebum and meibum. When 600 MHz NMR equipment is used in conjunction with highly sensitive cryogenically cooled probes, this method has adequate sensitivity, and for some applications, advantages over commonly used HPLC-evaporative light-scattering detection and mass spectrometry-based approaches. This method is shown to be useful for preclinical and clinical monitoring of the efficacy of sebum-reducing agents in animals and humans. In Syrian hamsters, 3% topical flutamide and 20 mg/kg oral isotretinoin reduced sterol esters by 18.7% and 30.0%, respectively, and reduced WEs by 32.9% and 31.8%, respectively, as measured in a punch biopsy of the ear. In a 72 patient clinical methodology study, the assay delivered reproducible and noninvasive measurements of WEs, cholesteryl esters, TGs, and squalene from Sebutape skin blots. The quantitative results of sebum analysis obtained by the NMR method correlate well with those obtained with HPLC-based approaches. This approach may be broadly applicable to cases in which fatty acid-independent quantification of lipid classes is desired.

Continuous flow configuration for total hydrocarbons index determination in soils by evaporative light scattering detection

A continuous flow configuration is proposed for the estimation of the hydrocarbon index in soils using for the first time evaporative light scattering detection (ELSD). The method is based on a membrane-enrichment of the hydrocarbons of interest (C 10–C 40), which are previously extracted from the soil matrix with a water:hexane mixture using a household microwave oven. The organic supernatant is cleaned-up through silica, evaporated and redissolved in a sodium dodecyl sulphate aqueous solution which is introduced in the loop of an injection valve. The sample, carried by an aqueous stream, passes through a continuous filtration unit fitted with a 1 μm pore size PTFE membrane, where the hydrocarbons are retained while the potential coextracted compounds are wasted. Quantitative elution of the C 10–C 40 fraction is accomplished by passing an acetone stream through the filter which drives the analytes to the ELSD system for analytical measurement. The detection limit was 1.8 μg ml −1, the linear range 5–25 μg ml −1 and the precision 4.3 %. The recoveries were between 93 and 97%. The proposed method was also evaluated by attenuated total reflexion-Fourier transform (ATR-FT-IR) analysis of the extracts.

Adaptation of an evaporative light-scattering detector to micro and capillary liquid chromatography and response assessment

A commercially available evaporative light-scattering detection (ELSD) system was adapted for micro and capillary LC. Therefore the various parameters involved in the droplet formation during the nebulization step in the ELSD system were studied. It was shown that the velocity term in the Nukiyama Tanasawa equation remains constant, leading to droplets of the same order of magnitude for narrow bore and capillary columns. Consequently, the ELSD modification was performed by decreasing the internal diameter of the effluent capillary tube in the nebulizer nozzle and by keeping its external diameter constant. Next, response curves for a conventional and the developed micro and capillary LC were compared as to investigate why a linear ELSD response is often obtained when used in micro or capillary LC. By splitting the flow rate post column, we showed that the nebulization process was not at the origin of the phenomenon. For ceramide III and tripalmitin, the response curves were found to be non-linear. However the curvature was less significant when the columns internal diameter decreased. Calculated particle size profiles for micro or capillary LC suggest that the particle entering the detection chamber are bigger than under conventional LC conditions. Last, triethylamine and formic acid were used to increase the response of the detector. The response enhancement, expected from previous studies, was established for the two lipids involved in this study.

Post-column addition as a method of controlling triacylglycerol response coefficient of an evaporative light scattering detector in liquid chromatography–evaporative light-scattering detection

The non-linear response is generally the main limitation to the general quantitative use of evaporative light-scattering detection (ELSD). In the particular case of triacylglycerol (TG) analysis, we present a preliminary paper dealing with the use of post-column additives as a means of monitoring the response of such a detector. As TG can form molecular association complexes (ligand–ligate associations) with either cholesterol, urea or silver nitrate, we report the influence of the concentration of each of these chemical compounds in the liquid phase directed towards the ELSD system. The results show that the response coefficient b of the calibration curve either decreases from 1.25–1.30 to 0.51 or increases from 1.25–1.30 to 1.78 according to the nature and concentration of post-column additive. The use of cholesterol as additive, at a discrete concentration, may lead to a linear response curve ( b=1), i.e. to the direct proportionality of ELSD response versus the TG concentration, making quantitative analysis of such solutes easier. On the other hand, to improve sensitivity, the addition of silver nitrate may be chosen for an increase in b value.