Separation ofCimicifuga racemosa triterpene glycosides by reversed phase high performance liquid chromatography and evaporative light scattering detection

Summary The main triterpene glycosides ofCimicifuga racemosa were separated by reversed phase HPLC, using a C-18 column, Evaporative Light Scattering (ELS) detection and a grient system consisting of water, acetonitrile and reagent alcohol. Within 35 min three main glycosides could be separated and quantified in the methanolic root extract with detection limits of 10.5, 15.6 and 31.6 μg·mL⁻¹ respectively. The method was successfully used, to analyzed differentCimicifuga racemosa market products, as well as to distinguish between otherCimicifuga samples from China.     

Interference of chitosan in glucose analysis by high-performance liquid chromatography with evaporative light scattering detection

The purpose of this work was to quantify glucose in aqueous solutions containing chitosan by high-performance liquid chromatography (HPLC) with evaporative light scattering detection (ELSD). Chitosan is a natural compound that is used alone or as an additive in several formulations. Microencapsulation of bioactive compounds such as glucose, by means of chitosan, is being explored, but difficulties arise when glucose needs to be determined in the presence of chitosan. HPLC is the technique most commonly used for glucose analysis, and ELSD may offer advantages (e.g. sensitivity and the possibility of operating in gradient mode) compared with other detectors. The influence of chitosan in the analysis of glucose by HPLC with ELSD was investigated at different pH values of the aqueous solutions. Isocratic elution with an acetonitrile/water mixture (80:20, v/v) and water washing between runs were the best options to avoid the mucoadhesive properties of chitosan, which are responsible for column degradation and variability of the retention time of glucose. The developed methodology was considered completely adequate for rapid glucose analysis in aqueous solutions with low pH (< 3), in the presence of chitosan.     

Fast and direct quantification of underivatized muscone by ultra performance liquid chromatography coupled with evaporative light scattering detection

A new reversed phase ultra performance liquid chromatography coupled with evaporative light scattering detection is developed for the fast and direct quantification of underivatized muscone in precious herbal medicine musk. Separation of muscone was achieved on a Waters Acquity BEH C₁₈ (50 × 2.1 mm id, 1.7 μm) column. The runtime was as short as 5 min. The mode of evaporative light scattering detection was set at Impact On. The influence of evaporative light scattering detection condition on sensitivity was investigated. The optimized condition was: drift tube temperature at 30°C, gas flow rate 4.2 L/min. The method was validated with respect to the precision, sensitivity, accuracy, linearity, stability, and robustness were measured in this paper. The calibration curves showed good linear regression (r = 0.9914) within the test range. The recovery rate was 98.6%. The limit of detection for muscone was 2.0 ng. The validated method was rapid, simple, reproducible, and convenient for the quantification of muscone in musk and the related products.     

Separation of monosaccharides by hydrophilic interaction chromatography with evaporative light scattering detection

Hydrophilic interaction liquid chromatography (HILIC) was used to separate monosaccharides that are common in N-linked oligosaccharides in glycoproteins and other compounds. A TSKgel Amide-80 column was eluted with 82% acetonitrile, in 5 mM ammonium formate (pH 5.5). Column temperature was 60 degrees C and evaporative light scattering was used for detection (ELSD). With this method, L-fucose, D-galactose, D-mannose, N-acetyl-D-glucosamine, N-acetylneuraminic acid, and D-glucuronic acid were separated, with detection limits of 0.3-0.5 microg for each monosaccharide, and intermediate precisions were 3-6% RSD (n=6).     

Simultaneous quantitation of three major triterpenoid glycosides in Centella asiatica extracts by high performance liquid chromatography with evaporative light scattering detection

A high-performance liquid chromatography method with evaporative light scattering detection was established for simultaneous determination of three major triterpenoid glycosides, i.e. asiaticoside, madecassoside and asiaticoside-B, in Centella asiatica extracts. The optimal chromatographic conditions were achieved on a COSMOSIL 5C(18)-MS-II column by constant elution with water (0.01% trifluoroacetic acid, v/v) and acetonitrile (1.0% methyl tert-butyl ether, 0.01% trifluoroacetic acid, v/v) (78:22) as mobile phase at a flow rate of 1.0 mL/min; the column temperature was 30 degrees C. The evaporative light scattering detector was set at an evaporating temperature of 40 degrees C and nitrogen gas pressure of 3.5 bar. The validation of the method included tests of linearity, sensitivity, precision, repeatability, stability and accuracy. All calibration curves showed good linear regression (r(2) > 0.9993) within test ranges. The established method showed good precision and accuracy with overall intra-day and inter-day variations of 1.73-3.06 and 3.89%-4.92%, respectively, and overall recoveries of 97.63-99.39% for the three compounds analyzed. The method developed was successfully applied to quantify the main triterpenoid glycosides in Centella asiatica extracts from different companies.     

Anomalies in evaporative light scattering detection

A two-dimensional (2-D) “heart-cutting” HPLC system was used to fractionate oligostyrenes into the respective diastereoisomers. For samples of known composition, the response of an ultraviolet (UV) absorbance detector followed the anticipated pattern. The response of an evaporative light-scattering (ELSD) detector on the other hand indicated quite different concentrations for the two diastereoisomers, relative to what was anticipated and what was indicated by the UV detector. Whereas approximately the same concentration was indicated by UV, ELSD in some cases indicated no detection of the later eluting isomer. The magnitude of the errors depended on both the molecular weight and the tacticity of the diastereomers. These anomalies appear to be an artifact of power transform functions imbedded within the firmware processor of the ELSD, invisible to the user.     

Fingerprint analysis of Dioscorea nipponica by high-performance liquid chromatography with evaporative light scattering detection

High-performance liquid chromatographic method (HPLC) with evaporative light scattering detection (ELSD) coupled with microwave-assisted extraction (MAE) as an efficient sample preparation technique has been developed for fingerprint analysis of Dioscorea nipponica. The samples were separated with an Agilent C8 column using water (A) and acetonitrile (B) under gradient conditions (0-10 min, linear gradient 20-40% B; 10-12 min, linear gradient 40-42% B; 12-25 min, isocratic 42% B) as the mobile phase at a flow rate of 1 mL min⁻¹ within 22 min. The ELSD conditions were optimized at nebulizer-gas flow rate 2.7 L min⁻¹ and drift tube temperature 90 °C. Precision experiments showed relative standard deviation (R.S.D.) of peak area and retention time were better than 2.5%; inter-day and intra-day variabilities showed that R.S.D. was ranged from 0.78% to 4.74%. Limit of detection was less than 50 μg mL⁻¹ and limit of quantification was less than 80 μg mL⁻¹. Accuracy validation showed that average recovery was between 97.39% and 104.07%. The method was validated to achieve the satisfactory precision and recovery. Relative retention time and relative peak area were used to identify the common peaks for fingerprint analysis. There are nine common peaks in the fingerprint. The quality of seven batches of D. nipponica samples was evaluated to be qualified or unqualified by the parameters “difference” and “total difference” of common peaks. Furthermore, the contents of important medicinal compounds (dioscin, prodioscin and gracillin) in different batches of D. nipponica samples were determined simultaneously using the developed HPLC-ELSD method. The results indicated variation of the herb quality which might be related to different producing area, growing condition, climate, harvest time, drug processing and so on. The developed analytical procedure was proved to be a reliable and rapid method for the quality control of D. nipponica.     

Direct analysis of industrial oligoglycerols by liquid chromatography with evaporative light-scattering detection and mass spectrometry

Summary A liquid chromatographic method has been developed for analysis of industrial polyglycerols, precursors of polyglycerol fatty esters, which are non-ionic surfacetants. The method utilizes two complementary chromatographic systems: porous graphitic carbon and an aminopropyl polymer with an acetonitrile-water mixture as mobile phase. Detection of these non-UV absorbing compounds was effected by means of evaporative light-scattering detection. Their structures were determined by comparison of their retention with that of synthesized standards, and by mass spectrometry.     

Separation ofAstragalus saponins by reversed phase high performance liquid chromatography and evaporative light scattering detection

Summary A new HPLC method permitted the separation of 13 triterpene lycosides isolated from differentAstragalus species within 40 min. A water/acetonitrile gradient was used as eluent and 5 μm RP-18 material as stationary phase. By using an evaporative light scattering (ELS) detector, the main saponins ofA. membranaceus could be detected at levels as low as 20.0 μg·mL⁻¹. This method facilitated distinction of differentAstragalus species as well as the analyses of market products containingA. membranaceus. The results showed variations from 0.019 to 0.184% in the total saponin content of the market products.     

Isolation of ceramide fractions from skin sample by subcritical chromatography with packed silica and evaporative light scattering detection

Separative method of lipid classes from the stratum corneum was developed with packed silica and supercritical CO2 containing 10% of methanol at 15 degrees C, 15 MPa and 3 ml min(-1). The elution order of lipid classes was first esterified cholesterol, triglycerides, squalene co-eluted in a single peak, then free fatty acids, free cholesterol, ceramides and finally glycosylceramides. The ceramides were eluted in several fractions which depended on the number of hydroxyl groups in the molecule, i.e. more hydroxyl groups were contained in ceramides, more important was the retention. Moreover, the retention was not altered by the presence of carbon double bond and variation of the alkyl chain length. The ceramide response with the evaporative light scattering detector was improved by turning the influence of the solvent nature on the response to advantage. Therefore, addition of various solvents with or without triethylamine and formic acid were tested in post-column due to the incompatibility of such modifiers with silica stationary phase. Thereby the solvent conditions for the separation and the detection can be adjusted almost independently. The response was greatly increased by post-column addition of 1% (v/v) triethylamine and its equivalent amount of formic acid in dichloromethane introduced at 0.1 ml min(-1) into the mobile phase. This device had allowed the detection of 400 ng of ceramide with a S/N = 21, whereas no peak was observed in absence of the post-column addition. Finally, the method was applied to the treatment of skin sample which led to highly enriched ceramide fraction.     

Quantification and identification of bioactive metabolites from Kalopanacis Cortex by HPLC with evaporative light scattering detection and ESI quadrupole TOF MS

A method based on HPLC coupled with an evaporative light scattering detection and ESI quadrupole TOF MS was established for the quantification and identification of phenolics and triterpene saponins in Kalopanacis Cortex using a gradient elution of acetonitrile with 0.1% formic acid and water with 0.1% formic acid on an RP C₁₈ column (4.6 × 250 mm, 5 μm). Diverse validation parameters, such as the linearity, LOD and LOQ, accuracy, precision, repeatability, and stability, were successfully obtained. Additionally, the efficiencies of different extraction methods were compared. The developed method was applied for the quantitative analysis of twelve representative metabolites in 61 Kalopanacis Cortex samples. The quantitation results showed that coniferin, kalopanaxsaponin C, septemlosides II, III, C, and D exhibited distinct regional patterns in Kalopanacis Cortex samples. These six compounds including one new triterpene saponin show potential as marker compounds for evaluating the quality of Kalopanacis Cortex and the geographical variation in its chemical composition.     

Characterization of sodium laureth sulfate by reversed-phase liquid chromatography with evaporative light scattering detection and H nuclear magnetic resonance spectroscopy

A direct and effective method utilizing reversed-phase liquid chromatography combined with evaporative light scattering detection was developed to determine the relative ratio of different alkyl chain lengths, to quantify the average ethylene oxide (EO) level and to identify EO distribution in the presence of sodium laureth sulfates. A C8 bonded silica gel column and an acetonitrile–water gradient mobile phase containing ammonium acetate were used as the best stationary and mobile phase, respectively. The results were confirmed by nuclear magnetic resonance spectroscopy. The detection limit was 80 μg/mL and the calibration curve, i.e., the log–log plots (peak area vs. concentration), was linear in the working range of 80–4200 μg/mL with R² values of above 0.999 (in the case of 3 mol sodium laureth sulfates). Furthermore, the application of the chromatographic method to a commercial product without pretreatment was presented. The raw material was identified by fast atom bombardment mass spectrometry.     

离子对反相高效液相色谱-蒸发光散射检测法测定三乙膦酸铝的含量

建立了以正丁胺为离子对试剂的反相高效液相色谱分析三乙膦酸铝含量的方法。采用Symmetry Shield RP18色谱柱分离,以甲醇-0.5%正丁胺水溶液(冰乙酸调节pH 5.0)(体积比为8:92)为流动相,流速为0.8 mL/min,蒸发光散射检测器(ELSD)检测。在上述条件下,三乙膦酸铝与其主要杂质亚磷酸盐、硫酸盐可以获得分离。在100～1200 mg/L范围内,进样质量与峰面积的双对数值呈良好的线性关系。100 mg/L和1000 mg/L两种质量浓度添加水平的回收率分别为100.58%和99.53%,其相对标准偏差(RSD)分别为0.62%和0.49%。该方法简便快捷,为三乙膦酸铝的定量分析提供了更加有效可靠的方法。     

Simultaneous determination of seven triterpenoids and triterpenoid saponins in Folium llicis Purpureae by high performance liquid chromatography coupled with evaporative light scattering detection

A new HPLC coupled with evaporative light scattering detection method was established for the simultaneous determination of seven triterpenoids and triterpenoid saponins in Folium Ilicis Purpureae traditional Chinese medicinal herb derived from the leaf of Ilex purpurea, namely, 3-omicron-beta-D-glucopyranosyl (1-->2)-beta-D-[6-omicron-methyl-glucuronopyranosyl]-28-omicron-beta-D-glucopyranosyl oleanolic acid (SQ-1), pedunculoside (SQ-2), 23-hydroxytormentic acid 28-omicron-beta-D-glucopyranoside (SQ-3), 23-hydroxytormentic acid (SQ-4), rutundic acid (SQ-5), ilexoside B (SQ-6), and ursolic acid (SQ-7). The optimal chromatographic conditions were achieved on a C18 column with a linear gradient elution of mobile phase A: deionized water-isopropyl alcohol-THF-acetic acid (90:10:6:1, v/v) and B: methanol-isopropyl alcohol-THF-acetic acid (90:10:6:1, v/v) at the flow rate of 1.0 mL/min; temperature for drift tube was set at 98degreesC and nitrogen flow rate was 2.8 L/min. All calibration curves of the seven compounds showed good linear regression (r2 > 0.995) within test ranges. The developed method has good repeatability for quantitation of all analytes interested with overall intraday and interday variation of less than 4.1%. The LOD (S/N = 3) and LOQ (S/N = 10) were less than 0.13 and 0.26 microg/mL, respectively, for all analytes. The established method was successfully used to evaluate the quality of Folium Ilicis Purpureae samples of different collections.     

高效液相色谱法测定烟草料液中的糖

研究了用蒸发光散射检测器检测，高效液相色谱法测定烟草料液中糖的方法。料液中的糖用固相萃取预分离，然后以Waters carbohydrate高效糖柱为固定相，V（乙腈）：V（水）=70：30作为流动相分离，蒸发光散射检测器检测；样品中鼠李糖、木糖、阿拉伯糖、果糖、甘露糖、葡萄糖、蔗糖、麦芽糖8种糖的加标回收率分别为：97．0％、95．6％、102％、102．1％、95．0％、101．8％、102．6％、97．8％；线性范围分别为：鼠李糖、果糖、葡萄糖、蔗糖0．1～20pg，木糖、阿拉伯糖、甘露糖、麦芽糖0．2～25μg。相对标准偏差均小于3．2％。方法的检出限达：鼠李糖20ng、木糖26ng、阿拉伯糖28ng、果糖14ng、甘露糖20ng、葡萄糖10ng、蔗糖12ng、麦芽糖15ng，用该方法测定了烟草料液中的糖。     

Simultaneous determination of multiple platycosides with a single reference standard in Platycodi Radix by high‐performance liquid chromatography coupled with evaporative light scattering detection

A traditional external standard method using HPLC coupled with evaporative light scattering detection has been developed for fast and accurate determination of seven platycosides in Platycodi Radix. However, inevitable difficulties in reference standards preparation process, which are quite costly and time consuming, have made its application limited. To avoid this inconvenience, a simultaneous determination of multiple components with a single reference standard strategy, which could be realized by calibrating the standard curve with internal standard and response factors, was introduced to the HPLC coupled with evaporative light scattering detection method. This is the first time that an incorporation of these two methods has been realized. Among seven ingredients, platycodin D was selected as the internal standard for its relatively easy preparation and low cost. Moreover, according to the investigation on concentration‐dependent effects over response factors and robustness test, platycoside E, deapioplatycodin D, platycodin D, and polygalacin D₂ were chosen to be the indicators for this novel method. The present method has not shown statistically significant differences with a traditional external standard method as verified sample analysis by the F‐test (p = 95%, n = 6).     

Preparative separation of dioscin derivatives from Dioscorea villosa by centrifugal partition chromatography coupled with evaporative light scattering detection

Dioscin derivatives from the Dioscorea villosa roots were isolated and purified by centrifugal partition chromatography coupled with evaporative light scattering detection. The two-phase solvent system composed of chloroform-methanol-isopropanol-water (7:6:1:4, v/v/v/v) was used to obtain prosapogenin A of dioscin (1, 11.1 mg), dioscin (2, 8.9 mg), deltonin (3, 29.2 mg) and diosgenin 3-O-[alpha-L-rhamnopyranosyl(1 --> 2)]-[beta-D-glucopyranosyl(1 --> 3)-beta-D-glucopyranosyl(1 --> 4)]-beta-D-glucopyranoside (4, 6.2 mg) from 300 mg of saponin-rich extract from the roots of D. villosa. The purities of 1, 2, 3 and 4 were determined to be 98.9, 97.4, 99.2 and 99.5%, respectively. Their chemical structures were determined by spectroscopic data of ESI-MS, 1H NMR and 13C NMR and comparing with those of previously reported values.     

液相色谱-串联质谱法测定海参和海胆中的单唾液酸神经节苷脂

建立了液相色谱-四极杆串联质谱法定量检测海参和海胆中单唾液酸神经节苷脂的分析方法。采用Svennerholm法从海胆或海参样品中提取神经节苷脂,经C8固相萃取柱净化,采用APS-2 NH₂柱(150 mm×2.1 mm, 3 μm),以乙腈和50 mmol/L乙酸铵溶液(pH 5.6)为流动相,梯度洗脱。样品中每种成分的定量在多反应监测模式下进行。该方法具有极高的灵敏度,定量限可低至纳克级。非硫酸酯化单唾液酸神经节苷脂(NMG)和硫酸酯化单唾液酸神经节苷脂(SMG)在1～40 ng进样量范围内呈现良好的线性关系;定量结果显示所测海参样品中美国红参的NMG含量最高,海胆样品中紫海胆的SMG含量最高;海胆中总的单唾液酸神经节苷脂含量(4.30～6.40 mg/g)明显高于各海参样品(8～131 μg/g)。该方法稳定可靠,适合海胆和海参中微量单唾液酸神经节苷脂的定量分析。     

Uncertainty in the determination of glucose in aqueous solutions by high‐performance liquid chromatography with evaporative light scattering detection

The determination of glucose and other carbohydrates is the most widespread chemical analysis that is performed within the industries of food, beverage, forage, biomass, pulp and paper, pharmaceuticals among others. Besides that, sugar refineries need to control their products, by‐products and effluents, and furthermore, glucose in the sucrose refining process, is considered an impurity, which shall be controlled. Being HPLC the most currently instrumental technique used for glucose analysis, the evaporative light scattering detector (ELSD) offers advantages (sensitivity, possibility for operating in gradient mode) over the also used refractive index detector. In this work, an HPLC‐ELSD methodology was optimised and validated, aiming the estimate of the uncertainty associated with the results at low levels of concentration of glucose to be measured. Linearity of the response was obtained in the range of glucose concentrations from 20 to 300 mg/L, with an analysis time of 10 min. The global uncertainty was estimated accordingly to the bottom‐up approach used by Eurachem. It was 13% on average for concentrations from 100 to 300 mg/L. For lower concentrations, uncertainty increased significantly up to 30% in the vicinity of the LOD of the method.     

Separation and identification of oligomeric ethyl silicates by liquid chromatography with electrospray ionization mass spectrometry

Reversed‐phase liquid chromatography coupled with electrospray ionization mass spectrometry was used to study the molecular structures of components and molar mass distributions in ethyl silicate‐40, a versatile liquid precursor for silicon‐based materials. Identity testing by standard spectroscopic techniques showed that a commercial sample of ethyl silicate‐40 was composed of linear/branched ethoxysiloxane oligomers with the silicon atoms ranging from 2 to 12 together with minor monocyclic species. Analysis of the sample by liquid chromatography coupled with evaporative light scattering detection resulted in an elution profile consisting of a series of peak clusters. Peak identification showed that the linear/branched homologous series of oligomers were eluted in the order of increasing number of silicon atoms in the molecules and the time duration (width) of the resulting peak clusters increased in the same fashion corresponding to increasing number of geometric isomers. In addition, small amounts of monocyclic oligomers present in the sample were found to be less retained than each linear/branched counterpart. Finally, the molar mass distribution parameters for ethyl silicate‐40 determined by the developed method were in good agreement with the literature values. Overall, this work demonstrates that reversed‐phase liquid chromatography coupled with electrospray ionization mass spectrometry is an indispensable tool for the comprehensive characterization of complex mixtures of this type.