Evaporative Light Scattering Research Articles

Improved methods for extraction and quantification of resin and rubber from guayule

Guayule, a shrub native to the Chihuahuan desert, is a natural source of high quality, hypoallergenic rubber. Unlike rubber trees that produce rubber in laticifers, the rubber in guayule is produced in parenchyma cells of the bark tissue of stems and roots. Consequently, guayule tissue must be mechanically broken before the rubber can be extracted and analyzed. Since rubber extraction and analysis is time-consuming, progress towards increasing the rubber content of guayule through breeding or better cultivation practices has been limited by the slow rate of sample processing. To address the need for faster and more efficient sample throughput, conditions were optimized for automated extraction of dried guayule tissue using accelerated solvent extraction (ASE) and rapid methods were developed to replace gravimetric determination of resin and rubber content. For resin analysis, ultraviolet absorbance was used to determine resin concentration after ASE of the tissue with acetone or acetonitrile. For rubber analysis, evaporative light scattering (ELS) was used to determine the amount of rubber recovered after ASE of the tissue with cyclohexane. Extraction of guayule tissue with high latex rubber content verified that the amounts of resin and rubber determined by these methods were similar to the amounts determined gravimetrically. Since these methods automate extraction and increase the speed of resin and rubber quantification, they could be used in combination with ASE to increase the throughput and efficiency of guayule evaluation in germplasm enhancement and agronomic improvement programs.

Abnormalities in Lung Surfactant in Horses Clinically Affected with Recurrent Airway Obstruction (RAO)

Abnormalities in lung surfactant are well described in human respiratory diseases including asthma, but are poorly described in horses. Lung surfactant is abnormal in horses with clinical signs of recurrent airway obstruction (RAO). Six healthy horses and 5 horses with RAO. Bronchoalveolar lavage fluid (BALF) was obtained from all horses by standard procedures. Cell-free BALF was separated into crude surfactant pellets (CSP) and supernatant via ultracentrifugation. Phospholipid and protein content was analyzed from both of these fractions. Phospholipid composition of CSP was determined using high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was measured with a pulsating bubble surfactometer. Compared with healthy horses, surfactant from RAO-affected horses was characterized by significantly decreased phospholipid content in total surfactant (median; range: 23.2; 14.7-62.2 microg/mL BALF versus 172; 111-267 microg/mL BALF, P = .0062) and CSP (20.2; 6.4-48.9 microL/mL BALF versus 155; 94.4-248 microg/mL BALF, P = .0062), and a significantly lower percentage of phosphatidylglycerol (PG) (4.5; 3.6-5.6% versus 6.6; 4.1-7.6%, P = .028). Furthermore, the ratio between the percentages of phosphatidylcholine and PG was significantly higher in RAO-affected horses than in healthy horses (20.9; 16.6: 25.9 versus 13.9; 11.8-22.8, P = .045). This study demonstrates that surfactant from RAO-affected horses is abnormal. Further studies are needed to determine if these abnormalities are related to an increased tendency for bronchoconstriction and to a decreased ability to clear airway mucus in RAO-affected horses.

Preparative Separation and Purification of β-Caryophyllene from Leaf Oil of Vitex negundo L. var. heterophylla (Franch.) Rehd. by High Speed Countercurrent Chromatography

High speed countercurrent chromatography (HSCCC) coupled with an evaporative light scatter detector (ELSD) was successfully applied to preparative isolation and purification of β-caryophyllene from essential oil of Vetix negundo L. var. heterophylla (Franch.) Rehd. Using either n-hexane-chloroform-acetonitrile (6:2:5) or n-hexane-dichloromethane-acetonitrile (10:3:7) as the two-phase solvent system, β-caryophyllene was separated in one run from the crude essential oil by HSCCC. However, the application of n-hexane-dichloromethane-acetonitrile (10:3:7) resulted in a higher purity and recovery of β-caryophyllene. About 85 mg of β-caryophyllene representing 97.3% by GC, was yielded from 600 mg crude essential oil when eluted with the lower phase at a flow rate of 1.5 mL.min−1.

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.

Analytical Methods for Determination of Counter-ions in Pharmaceutical Salts

Inorganic ions as well as organic acids or bases are frequently employed as counter-ions of ionizable drug substances. Therefore, analytical methods are required to confirm production of the correct salt form and to quantify the level of the counter-ion present in each batch of drug substances manufactured. Several options exist for determination of counter-ions in pharmaceutical salts and will be discussed in this review. Ion chromatography (IC) with conductivity detection is probably the most frequently employed method for ion determination. Other techniques such as capillary electrophoresis (CE) represent an alternative to IC for determination of counter-ions in pharmaceutical salts. Due to the lack of chromophores, detection of small ions in CE is typically performed using indirect UV detection. Hydrophilic interaction chromatography (HILIC) with evaporative light-scattering (ELSD) or refractive index detection can also be used for determination of non-UV absorbing ions. Each technique will be described and their advantages highlighted. Current and future trends in measuring counter-ions in pharmaceutical salts will also be discussed. Keywords: Counter-ions, Pharmaceutical, Ion chromatography, Capillary electrophoresis, HILIC, Mixed-mode chromatography

Chromatographic fingerprint development for herbal extracts: A screening and optimization methodology on monolithic columns

Herbs are used worldwide for preventive and therapeutic purposes. Therefore, identification and quality control of products of vegetal origin is required. Fingerprint chromatography is accepted by the World Health Organization as an identification and quality evaluation technique for medicinal herbs. In fingerprint development, the first step is to create general conditions maximizing the peak capacity. Four herbs, Liquorice, Cascara, Curcuma and Artichoke, were examined using different experimental conditions in order to propose a methodology to develop fingerprint chromatograms in high-performance liquid chromatography (HPLC) with ultra-violet (UV) and evaporative light scattering (ELS) detection. The methodology comprised a screening and an optimization phase. Monolithic C18 columns were used as stationary phases. Several organic modifiers were tested to define a gradient elution system with an optimal selectivity. From the screening phase, linear gradients of 5–60%, 5–54%, 5–95% and 5–95% acetonitrile for Liquorice, Cascara, Curcuma and Artichoke, respectively, were selected. The ELS detection did not provide additional information to the UV detection. The optimization phase selected the best experimental conditions for wavelength, column length, flow rate and slope of the gradient for the best modifier determined at the end of the screening. For the four examined herbs 254 nm as detection wavelength, 300 mm column length, 2 ml/min flow rate and a gradient time of 50 min were defined as the best conditions. If these latter unique conditions are confirmed for other case studies, the actual strategy even might be simplified by reducing the optimization stage.

Size Exclusion Chromatography for the Unambiguous Detection of Aliphatics in Fractions from Petroleum Vacuum Residues, Coal Liquids, and Standard Materials, in the Presence of Aromatics

A method has been developed using size exclusion chromatography (SEC) in heptane eluent that can detect aliphatics unambiguously without fractionation to remove aromatics. Spherical molecules such as colloidal silicas elute at the exclusion limit, while alkanes up to C{sub 50} elute through the porosity of the column. Detection of aliphatics was defined by use of an evaporative light scattering (ELS) detector with the simultaneous absence of UV absorbance at 300 nm. Alkanes smaller than C{sub 12} were not detected because the conditions of operation of the ELS caused their evaporation. All aromatics eluted after the permeation limit of about 25 min and were not detected until well after 45 min by their UV absorbance. The SEC method was applied to petroleum vacuum residues and coal liquids, and their fractions were soluble in pentane or heptane. High-temperature (HT) GC-MS confirmed the presence of alkanes in the pentane- and heptane-soluble fractions of petroleum vacuum residues, but did not elute any of the aromatics known to be present from SEC. Alkanes were examined in pentane-soluble fractions of a coal digest and a low-temperature coal tar; alkanes up to C{sub 40} were detected in the low-temperature tar and, although present in the digest, weremore » masked by aromatics. No alkanes were detected by either SEC or HT GC-MS in fractions from a coal tar pitch. Aromatics in coal liquids and one petroleum residue were also examined by SEC using NMP as eluent and by UV fluorescence spectroscopy. The SEC method will find application to pentane- and heptane-soluble fractions of petroleum liquids and coal liquids where the alkanes are concentrated relative to the more abundant aromatics. 43 refs., 10 figs., 2 tabs.« less

Lung Surfactant Function and Composition in Neonatal Foals and Adult Horses

Lung surfactant function and composition are varied and adapted to the specific respiratory physiology of all mammalian species. Lung surfactant function and composition are different in neonatal foals as compared to adult horses. Six adult horses, 7 term foals (<24 hours old), and 4 premature foals were used. Animals were part of the Auburn University teaching herd except for 3 client-owned premature foals. Bronchoalveolar lavage fluid (BALF) was obtained from all animals. Ultracentrifugation of cell-free BALF separated surfactant into crude surfactant pellets (CSP) and supernatant. Both fractions were analyzed for phospholipid and protein content with the Bartlett and bicinchoninic acid method, respectively. Phospholipid composition of the CSP was determined by using high-performance liquid chromatography with an evaporative light scatter detector. Surface tension of the CSP was measured with a pulsating bubble surfactometer. Results from term foals (<24 hours old) were compared statistically to those from adult horses. Values of P < .05 were considered significant. BALF phospholipid content was similar between adult horses and term foals, but BALF protein content was significantly decreased in term foals. Phosphatidylglycerol was significantly decreased, phosphatidylinositol was significantly increased, and the minimum surface tension was significantly increased in the CSP from term foals compared to adult horses. Surface tension and phospholipid composition of surfactant in neonatal foals are significantly different compared to adult horses. These changes may influence biophysical and immunologic functions of surfactant.

A 21st Century HPLC Workflow for Process R&amp;D

A unique high-performance liquid chromatographic (HPLC) workflow specifically designed for the rigors of process development has been developed. A key feature of the workflow is the creation of an HPLC software–hardware platform designed to automatically and systematically screen samples using a matrix of columns and eluents to aggressively search for impurities. The workflow platform was assembled from commercial hardware components and both custom and commercial HPLC software. The platform can be used to challenge existing HPLC methods or to develop new methods. The screening conditions are complementary to each other, and are useful to assess the complexity of a sample and to chromatographically resolve impurities that may coelute using any single method. The workflow has been designed to support several different modes of HPLC, and can be used with absorption detection, photodiode array spectrometers, evaporative light scattering (ELS) devices, and mass spectrometric (MS) detection. The custom software interface contains a data-viewing feature to simplify analysis of results. The platform is designed to be used by process scientists, and the same simple user-interface is used to control analytical HPLC, LC–MS, and preparative HPLC. Three real world examples are provided to illustrate the utility of the platform to rigorously assess the complexity of samples and to develop new and improved HPLC methods. (JALA 2005;10:381–93)

Determination of surface-bound hydroxypropylcellulose (HPC) on drug particles in colloidal dispersions using size exclusion chromatography: A comparison of ELS and RI detection

Evaporative light scattering (ELS) and refractive index (RI) detection methods were evaluated for the determination of surface-bound hydroxypropylcellulose (HPC) on drug particles in colloidal dispersions. Size exclusion chromatography (SEC) was used to separate HPC from other components of the dispersions. The instrumental parameters of the ELS detector were optimized to obtain maximum peak intensity, adequate peak shape and minimal baseline noise by varying the mobile phase flow rate, nebulizer temperature, and evaporation temperature. The chromatographic method was validated using both detectors. The ELS detector response exhibited second order polynomial and linear double logarithmic correlation with concentration over a 10–300% range while the RI response was linear. The double logarithmic correlation simplified the calculation compared to using the polynomial fit, and it provided more accurate results compared to the linear fit approach. Total HPC was obtained by solubilizing all components of the dispersion and analyzing for HPC. Non-bound HPC was obtained by ultracentrifuging the dispersion and analyzing the supernatant for HPC concentration. Analysis for total- and non-bound HPC in a representative colloidal dispersion gave method precisions with R.S.D.s of 2.5 and 2.2% for ELS, and 4.5 and 2.4% for RI ( n = 4). HPC bound to the surface of the drug particles was determined by difference: % bound HPC = 100% − % non-bound HPC. Resultant % bound HPC values ranged from 22.1 to 25.4% of available HPC. Both ELS and RI are satisfactory detection techniques for HPC quantitation and for determination of the proportion of HPC bound to drug colloid particles, and the assay results are comparable.

Analytical and Biological Evaluation of High Throughput Screen Actives Using Evaporative Light Scattering, Chemiluminescent Nitrogen Detection, and Accurate Mass LC-MS-MS

In this investigation the utility of evaporative light scattering detection (ELSD) combined with HPLC-MS was demonstrated as a key component of a bioassay-guided fractionation, or "biofractionation" technique, for the evaluation of high throughput screen actives. ELSD provided on-line analyte mass information that was critical for the classification of the samples. Chemiluminescent nitrogen detection (CLND) was also evaluated for sample concentration estimation for nitrogen-containing compounds, and accurate mass LC-MS-MS analysis was employed for rapid structural confirmation and elucidation of components previously identified as active via biofractionation.

Development of a Ginkgo biloba fingerprint chromatogram with UV and evaporative light scattering detection and optimization of the evaporative light scattering detector operating conditions

A fingerprint chromatogram of a standardized Ginkgo biloba extract is developed on a monolithic silica column using a ternary gradient containing water, iso-propanol and tetrahydrofuran. For the detection, UV and evaporative light scattering (ELS) detectors are used, the latter allowing detection of the poor UV absorbing compounds as ginkgolides (A–C and J) and bilobalide in the extract. The complementary information between the UV and ELS fingerprint is evaluated. The ELS detector used in this study can operate in an impactor ‘on’ or ‘off’ mode. For each mode, the operating conditions such as the nebulizing gas flow rate, the drift tube temperature and the gain are optimized by use of three-level screening designs to obtain the best signal-to-noise (S/N) ratio in the final ELS fingerprint chromatogram. In both impactor modes, very similar S/N ratios are obtained for the nominal levels of the design. However, optimization of the operating conditions resulted, for both impactor modes, in a significant increase in S/N ratios compared to the initial evaluated conditions, obtained from the detector software.

Analysis of Phospho- and Sphingolipids in Dairy Products by a New HPLC Method

Dairy phospho- and sphingolipids are gaining interest due to their nutritional and technological properties. A new HPLC method, using an evaporative laser light-scattering detector, was developed, which enabled excellent separation of glucosylceramide, lactosylceramide, phosphatidic acid, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, sphingomyelin, and lysophosphatidylcholine in less than 21min, including the regeneration of the column. No loss of column performance was observed after 1500 runs because an acid buffer was used. The output signal of the evaporative laser light scattering detector was highly dependent of the flow of the carrier gas and the temperature of the nebulizer, and was maximized by means of a response surface experimental design. Finally, raw milk, cream, butter, buttermilk, Cheddar whey, quarg, and Cheddar cheese were analyzed for their polar lipid content. The absolute values varied substantially (0.018 to 0.181 g/100g of product). Significant differences were found in the relative content of each polar lipid class among the analyzed products.

Separation of Polybutylene Glycols Widely Differing in Molecular Weight on Different Stationary Phase by Gradient RP-HPLC and Detection by Evaporative Light Scattering

Polybutylene glycols (PBG’s) substantially differing in molecular weight (MW), i.e., PBG 650, PBG 1000, PBG 2000 and PBG 3000 were subjected to separation on monolithic, i.e., Chromolith SpeedRod RP-18 and Chromolith Performance RP-18, polymeric, i.e., Polymer C18 and silica-based, i.e., Nucleosil 5C4 chromatographic supports. Different eluent systems each consisting of a ternary gradient built up from acetonitrile, tetrahydrofuran and water were applied and monitoring of signal responses was accomplished by evaporative light scattering detection (ELSD). Fine-tuning of the gradient profiles for the different PBG’s yields optimum separation of the individual oligomers of the low MW samples PBG 650 and PBG 1000 and no marked differences seem to occur on the four stationary phases used within the study. However in contrast, pronounced deviations between them were observable when applied for separation of PBG 2000 and PBG 3000. Surprisingly in the latter case the Nucleosil 5C4 support proved to be better suited for separation of sample constituents of higher MW. Although better resolution into individual oligomers, in particular of PBG 2000 and PBG 3000, was achieved on both Chromolith SpeedRod RP-18 and Chromolith Performance RP-18 when compared to the Polymer C18 support, the good separation efficiency of the latter support is nevertheless quite remarkable making this material an additional valuable alternative for chromatographic fingerprinting at least for PBG 650 and PBG 1000.

Ammonium hydrogencarbonate, an excellent buffer for the analysis of basic drugs by liquid chromatography–mass spectrometry at high pH

Ammonium hydrogencarbonate buffer has been found to be especially useful for high-pH HPLC analysis of samples from both combinatorial and medicinal chemistry sources. Satisfactory results were obtained by the standard diode array, evaporative light-scattering, and MS detection by using this buffer at a concentration of 10 m M. From a practical standpoint, ammonium hydrogencarbonate is an ideal buffer for chromatographers since it provides excellent chromatographic behaviour and reproducible separation. In addition to this, its volatility makes it an essential tool for rapid LC–MS product identification. Ammonium hydrogencarbonate was tested for a number of drug-like compounds analysed as mixtures, and data obtained were compared to those from the classical and MS-friendly buffers widely used by chromatographers: trifluoroacetic and formic acids. The results of this study revealed the suitability of this buffer for routine HPLC application in research laboratories.

IRDye78 conjugates for near-infrared fluorescence imaging.

The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.

IRDye78 Conjugates for Near-Infrared Fluorescence Imaging

The detection of human malignancies by near-infrared (NIR) fluorescence will require the conjugation of cancer-specific ligands to NIR fluorophores that have optimal photoproperties and pharmacokinetics. IRDye78, a tetra-sulfonated heptamethine indocyanine NIR fluorophore, meets most of the criteria for an in vivo imaging agent, and is available as an N-hydroxysuccinimide ester for conjugation to low-molecular-weight ligands. However, IRDye78 has a high charge-to-mass ratio, complicating purification of conjugates. It also has a potentially labile linkage between fluorophore and ligand. We have developed an ion-pairing purification strategy for IRDye78 that can be performed with a standard C18 column under neutral conditions, thus preserving the stability of fluorophore, ligand, and conjugate. By employing parallel evaporative light scatter and absorbance detectors, all reactants and products are identified, and conjugate purity is maximized. We describe reversible and irreversible conversions of IRDye78 that can occur during sample purification, and describe methods for preserving conjugate stability. Using seven ligands, spanning several classes of small molecules and peptides (neutral, charged, and/or hydrophobic), we illustrate the robustness of these methods, and confirm that IRDye78 conjugates so purified retain bioactivity and permit NIR fluorescence imaging of specific targets.

Separation and identification of phosphatidylserine molecular species using reversed-phase high-performance liquid chromatography with evaporative light scattering and mass spectrometric detection

A reversed-phase HPLC method compatible with evaporative light scattering (ELS) and electrospray mass spectrometric (ES-MS) detection was developed for separation of phosphatidylserine (PS) molecular species. The method was optimised for separation of three disaturated synthetic species: dipalmitoyl glycerophosphoserine, palmitoyl-stearoyl glycerophosphoserine and distearoyl glycerophosphoserine using isocratic elution with a mixture of 2-propanol, tetrahydrofuran and ammonium formate. Baseline separation was obtained on three different columns: one polystyrene/divinylbenzene (PS/DVB) column and two silica based C 18 and C 30 columns. The best chromatographic resolution was achieved with the C 30 column. The limit of detection for DPPS was 5 μg/ml ( S/ N=3) with ELS detection and 0.1 μg/ml ( S/N=3) with negative ion ES-MS in the single ion monitoring mode. Baseline separation of the five main species in a biological PS sample, bovine brain PS, was obtained with the PS/DVB column. Species identification was done by using the retention times of the intact PS species and their corresponding carboxylate anion fragments obtained by in-source fragmentation. Data have shown that individual PS species can be identified by their retention times using direct ELS detection in a mixture of disaturated PS species. However, for the bovine brain PS electrospray-MS detection was necessary for species identification due to the many possible fatty acid combinations in biological PS.

HPLC ANALYSIS OF GINSENOSIDES IN THE ROOTS OF ASIAN GINSENG (PANAX GINSENG) AND NORTH AMERICAN GINSENG (PANAX QUINQUEFOLIUS) WITH IN-LINE PHOTODIODE ARRAY AND EVAPORATIVE LIGHT SCATTERING DETECTION

In-line connected ultraviolet (UV) and evaporative light scattering (ELS) detection were compared in the reversed-phase high-performance liquid chromatographic (HPLC) analysis of ginsenosides in ginseng roots. Similar to all the QA/QC literature on ginseng and ginseng products, employing HPLC-UV, the UV detection showed a good linearity in the range of 100–2000 ng of ginsenosides, with minimum detectable concentration of 10 ng of ginsenosides on the column. In contrast, the sensitivity of the ELSD was found to be 5 times lower than that obtained with the UV, with minimum detectable concentration of 50 ng of ginsenosides on the column. Further, the calibration curve obtained with the ELSD was exponential linear in the range of 200–2000 ng of ginsenosides. Quantitative evaluation in terms of ginsenosides using both methods of detection showed similar results. But, in view of the better sensitivity and easier handling, the UV is still recommended for the routine analysis of ginseng samples.

Determination of Steroidal Saponins in Tribulus terrestris by Reversed-Phase High-Performance Liquid Chromatography and Evaporative Light Scattering Detection

This paper describes the first analytical method suitable for the determination of steroidal saponins in Tribulus terrestris. A separation by high performance liquid chromatography (HPLC) was achieved by using a reversed-phase (RP-18) column, evaporative light scattering (ELS) detection, and a water/acetonitrile gradient as the mobile phase. The marker compound, protodioscin, was detected at a concentration as low as 10.0 microg/mL. Several different samples of plant material were successfully analyzed, and depending on origin and plant part used for extraction, significant differences in the composition of the saponins were observed. The analysis of market products showed considerable variations of 0.17 to 6.49% in the protodioscin content.