Validation of HPLC‐UV method for determination of minor glycosides contained in Stevia rebaudiana Bertoni leaves

Leaves of Stevia rebaudiana contain glycosides with sweetness and biological activity. However besides the major glycosides, there are other glycosides within extracts that may contribute to its activity, and therefore it is important to quantify them. In this work, an isocratic HPLC method was validated for determination of dulcoside A, steviolbioside, rebaudioside C and rebaudioside B. An HPLC method was performed using a C₁₈ column (250 × 4.6 mm, particle size 5 µm) and a UV detector set at 210 nm. The mobile phase consisted of a 32:68 (v/v) mixture of acetonitrile and sodium phosphate buffer (10 mmol/L, pH 2.6), set to a flow rate of 1.0 mL/min. The calculated parameters were: sensitivity, linearity, limit of detection (LOD), limit of quantification (LOQ), accuracy and precision. The calibration curves were linear over the working range 25–150 µg/mL, with coefficient of correlation of ≥0.99 and coefficient of determination of ≥0.98. The LOD was 5.68–8.81 µg/mL, while the LOQ was 17.21–26.69 µg/mL. The percentage recoveries of fortified samples were 100 ± 10% and precision, relative standard deviation, was <10%. The method validation showed accuracy, linearity and precision; therefore this method can be applied for quantitative analysis of minor steviol glycosides in S. rebaudiana leaves. Copyright © 2014 John Wiley & Sons, Ltd.     

Tandem mass spectrometry: a convenient approach in the dosage of steviol glycosides in Stevia sweetened commercial food beverages

The use of sweeteners extracted from leaves of the plant species Stevia rebaudiana is increasing worldwide. They are recognized as generally recognized as safe by the US‐FDA and approved by EU‐European Food Safety Authority, with some recommendation on the daily dosage that should not interfere with glucose metabolism. The results presented here introduce an easy analytical approach for the identification and assay of Stevia sweeteners in commercially available soft drink, based on liquid chromatography coupled to tandem mass spectrometry, using a natural statin‐like molecule, Brutieridin, as internal standard. Copyright © 2017 John Wiley & Sons, Ltd.     

Endcapping of high performance liquid chromatography packings derived from silica gel

The effect of endcapping a reversed phase chromatographic packing material derived from silica gel depends on the degree of derivatization of the phase. The effect can be dramatic for phases with low loading. Whether an octadecylated silica gel has been endcapped or not can be ascertained by chromatographing the pair naphthalene/1-nitronaphthalene with a water/methanol eluent producing at least k' ≈ 10 for naphthalene. The ratio of the two k' values is 1.4 or higher for an endcapped material, while it is only 1.1 -1.2 for a non-endcapped phase. A similar approach fails to give conclusions of similar utility for octylated silica gels.     

Development of a high‐performance liquid chromatography procedure to identify known and detect novel C‐13 oligosaccharide moieties in diterpene glycosides from Stevia rebaudiana (Bertoni) Bertoni (Asteraceae): Structure elucidation of rebaudiosides V and W

As an aid for structure elucidation of new steviol glycosides, reversed‐phase C18 high‐performance liquid chromatography method was developed with several previously characterized diterpene glycosides, to identify known and detect novel aglycone‐C13 oligosaccharide moieties and indirectly identify C‐19 interlinkages. Elution order of several diterpene glycosides and their aglycone‐C13 oligosaccharide substituted with different sugar arrangements were also summarized. Comparison of the retention time of a product obtained after alkaline hydrolysis with the aglycone‐C‐13 portions of known compounds reported herein allowed us to deduce the exact positions of the sugars in the C‐13 oligosaccharide portion. The elution position of several steviol glycosides with an ent‐kaurene skeleton was helpful to describe an identification key. Two previously uncharacterized diterpene glycosides together with two known compounds were isolated from a commercial Stevia rebaudiana leaf extract. One was found to be 13‐[(2‐O‐β‐d ‐xylopyranosyl‐ 3‐O‐β‐d ‐glucopyranosyl‐β‐d ‐glucopyranosyl)oxy]ent‐kaur‐16‐en‐19‐oic acid‐(2‐O‐β‐d ‐glucopyranosyl‐β‐d ‐glucopyranosyl) ester (rebaudioside V), whereas the other was determined to be 13‐[(2‐O‐β‐d ‐xylopyranosyl‐ 3‐O‐β‐d ‐glucopyranosyl‐β‐d‐ glucopyranosyl)oxy]ent‐kaur‐16‐en‐19‐oic acid‐(2‐O‐α‐l ‐rhamnopyranosyl‐3‐O‐β‐d ‐glucopyranosyl‐β‐d ‐glucopyranosyl) ester (rebaudioside W). Previously reported compounds were isolated in gram quantities and identified as rebaudioside J and rebaudioside H. In addition, a C‐19 sugar‐free derivative was also prepared from rebaudioside H to afford rebaudioside H₁. Chemical structures were partially determined by the high‐performance liquid chromatography method and unambiguously characterized by using one‐dimensional and two‐dimensional nuclear magnetic resonance experiments.     

大黄素键合硅胶高效液相色谱柱的制备和应用

采用中间体法,先将大黄素配体与γ-[（2,3）-环氧丙氧]丙基三甲氧基硅烷（KH-560）偶联剂反应制备含配体的硅氧烷试剂,然后再与硅胶键合,最终制得大黄素键合硅胶液相色谱固定相（以下简称ESP）。通过红外光谱、元素分析和热重分析表征固定相的结构。以萘作为溶质探针,甲醇-水（60：40,v/v）为流动相,流速为0.8 mL/min,测得ESP柱的柱效。采用传统的反相C₁₈和苯基柱作参比,将ESP应用于系列中性、碱性和酸性芳香族化合物以及实际样品风油精的分离分析,并探讨相关的色谱分离机理。结果表明,配体大黄素被成功地键合到球形硅胶表面,测得配体键合量为0.23 mmol/g,ESP柱理论塔板数约为19874 N/m。ESP的偶联剂链和蒽醌环提供了疏水性的结构基础,大黄素配体还能为溶质提供π-π或p-π、电荷转移、氢键、偶极-偶极等作用点。多位点的协同作用使得ESP柱具有独特和优秀的色谱分离选择性,并且无需调节pH值,采用简单而廉价的甲醇-水流动相就能实现胺类、酚类等极性样品的基线分离,实验条件简单、方便。     

丁香酚键合硅胶液相色谱固定相的制备和色谱性能

采用固液相表面连续反应法,先将偶联剂γ-[(2,3)-环氧丙氧]丙基三甲氧基硅烷(KH-560)键合到球形硅胶表面,然后再将植物有效成分丁香酚与硅胶上的KH-560活性基团反应,合成了丁香酚键合硅胶液相色谱固定相(EGSP)。采用元素分析、热重分析和红外光谱对该固定相的结构进行了表征。以萘作为溶质探针,乙腈-水(35:65, v/v)为流动相,流速为0.8 mL/min,测得EGSP柱的柱效。以一系列的中性、碱性和酸性化合物为溶质探针,C₁₈柱和苯基柱作参比,对该固定相的色谱性能及保留机理进行了研究。结果表明,硅胶表面成功键合上了丁香酚配体,键合量为0.28 mmol/g, EGSP柱理论塔板数约为24707 N/m。该固定相不仅具有良好的反相色谱性能,同时由于配体结构中含有芳环、烯基和甲氧基,还能与溶质发生π-π电荷转移、偶极-偶极和氢键作用。与传统的反相C₁₈柱和苯基柱相比,EGSP在极性芳香族化合物的快速、简便分离中占优势。     

Group separation of alkyl-substituted aromatic hydrocarbons by high-performance liquid chromatography using perfluorocarbon modified silica gel

Summary The behaviour of perfluorocarbon modified silica gel has been investigated in HPLC for the group separation of alkyl-substituted aromatic hydrocarbons. Using dry alkanes as eluents, the influence of the substituted alkyl groups on the retention of mono- and polycyclic aromatics is very small. Therefore, aromatic hydrocarbons can be separated in order of their increasing -electron energy characterized by a linear correlation between ink and the resonance energy. The method is useful for the group separation of crude oil distillates into groups according to aromatic ring types.Presented at the 14th International Symposium on Chromatography London, September, 1982.     

Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata

In this study, a simple and reliable HPLC method for the qualitative and quantitative analysis of cardiac glycosides in Digitalis lanata Ehrh. raw material was developed and applied to healthy and phytoplasma-infected plants. The target analytes cover a broad range of secondary metabolites, including primary, secondary and tertiary glycosides and the corresponding aglycones. The sample preparation was carried out by sonication of the plant material with 70% (v/v) aqueous methanol at room temperature, followed by reversed-phase solid-phase extraction purification from interfering pigments. The HPLC analyses were performed on a Symmetry C₁₈ column (75 mm × 4.6 mm I.D., 3.5 μm), with a gradient elution composed of water and acetonitrile, at a flow rate of 1.0 mL/min. The column temperature was set at 20 °C and the photodiode array detector monitored the eluent at 220 nm. The method was validated with respect to ICH guidelines and the validation parameters were found to be highly satisfactory. The application of the method to the analysis of D. lanata leaves indicated that air-drying was the optimum method for raw material processing when compared with freeze-drying. The analysis of healthy and phytoplasma-infected plants demonstrated that the secondary metabolite mainly affected by the pathogen presence was lanatoside C (153.2 μg/100 mg versus 76.1 μg/100 mg). Considering the importance of D. lanata plant material as source of cardiac glycosides, the developed method can be considered suitable for the phytochemical analysis and for the quality assurance of D. lanata used for pharmaceutical purpose.     

Synthesis of monodispersed molecularly imprinted polymer particles for high-performance liquid chromatographic separation of cholesterol using templating polymerization in porous silica gel bound with cholesterol molecules on its surface

Monodispersed molecularly imprinted polymer particles selective for cholesterol were prepared by the copolymerization of styrene and divinylbenzene in the presence of template silica gel particles (particle size: 5 μm; pore size: 10 nm) functionalized with cholesterol on the surface, followed by dissolution of the cholesterol-bonded silica gel with a NaOH aqueous solution. Transmission and scanning electron micrographs of the molecularly imprinted polymer (MIP) particles revealed good monodispersity and porous structure. The MIP particles were packed into a high performance liquid chromatographic column, and its recognition ability of cholesterol was evaluated using cholesterol, cholesterol esters and fatty acid methyl esters by comparison with the non-imprinted polymer (NIP) particles prepared from styrene and divinylbenzene without cholesterol. The MIP particles showed a high affinity for cholesterol and cholesterol esters (K(MIP)'/K(NIP)' > 5.7).     

Phenylcarbamate derivatives of cellulose and amylose immobilized onto silica gel as chiral stationary phases for high-performance liquid chromatography

Three polysaccharide phenylcarbamate derivatives [cellulose 2,3-bis(3,5-dimethylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate)/(2-methacryloyloxyethylcarbamate), cellulose 2,3-bis(3,5-dichlorophenylcarbamate)-6-(3,5-dichlorophenylcarbamate)/(2-methacryloyloxyethylcarbamate), and amylose 2,3-bis(3, 5-dimethylphenylcarbamate)-6-(3,5-dimethylphenylcarbamate)/(2-methacryloyloxyethylcarbamate)] with vinyl groups were prepared and coated onto silica gel to immobilize them via radical copolymerization with 2,3-dimethylbutadiene. The copolymerization efficiently proceeded, and the coated polysaccharide derivatives were mostly immobilized on the surface of the silica gel. The immobilized polysaccharide derivatives showed high chiral recognition abilities similar to those of the corresponding coated polysaccharide derivatives. They could be used with an eluent containing chloroform, which dissolved the polysaccharide derivatives. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 4704–4710, 2004     

A new method for dissolution of silica gel in a pentafluoropropionyl fluoride—tertiary amine system

A new procedure for the preparation of amino complexes of silicon tetrafluoride was proposed. For this purpose, silica gel is dissolved in a fluoroanhydride—methanol system at room temperature followed by the precipitation of SiF₄ solvate that was formed with tertiary amine (pyridine, 1,10-phenanthroline, 2,2′-bipyridyl). The yields of the formed complexes are 69–83%. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 421–423, March, 2007.     

A reversed-phase high-performance liquid chromatography method with pulsed amperometric detection for the determination of glycosides

We have developed a reversed-phase high-performance liquid chromatography pulsed amperometric detection (RP-HPLC-PAD) method for the determination of glycosides. It is sensitive, repeatable, and selective without the pretreatment step. Ginsenosides were separated completely in 50 min using an water-acetonitrile gradient as the eluent and detected by PAD under NaOH alkaline conditions. The ginsenoside detection limit (S/N=3) was 0.02-0.07 ng and the quantification limit (S/N=10) was 0.1-0.2 ng. The coefficient of linear regression was 0.9984-0.9998 for concentrations between 1 and 50 microg/mL. The intra- and inter-day precision (RSD) was less than 6.35% in Ginseng Radix and Shy-jiun-tzyy-tang extracts. The average recoveries from Ginseng Radix and Shy-jiun-tzyy-tang extracts were 98.19-105.45% and 96.89-102.22%, respectively.     

Physico-chemical applications of liquid chromatography II. Investigations of the surface properties of chemically modified silica gels and of the adsorption of cardiac glycosides from solutions

Summary The chemical modification of silica surface using dichlorosilanes with methyl and phenyl groups and triethylamine as donor in the liquid phase was investigated. This modification leads to a dense coverage of the silica surface by the organic functional groups. Liquid chromatography permits the determination of the thermodinamic characteristics of intermolecular interactions of different molecules during adsorption from solutions on the modified surface of the silica. Silica gels modified with dichlorosilanes containing phenyl groups, possess high selectivity () and capacity (k) for the cardiac glycosides G-strophanthin, K-strophanthoside, K-strophanthin- and cymarin using water-alcohol mixture as the mobile phase, and the selectivity and capacity is much higher than observed on silica gels modified with methyl groups only.     

New method for development of carbon clad silica phases for liquid chromatography: Part I. Preparation of carbon phases

Owing to its combination of unique selectivity and mechanical strength, commercial carbon clad zirconia (C/ZrO₂) has been widely used for many applications, including fast two-dimensional liquid chromatography (2DLC). However, the low surface area available (only 20–30 m²/g for commercial porous ZrO₂) limits its retentivity. We have recently addressed this limitation by developing a carbon phase coated on the high surface area of HPLC grade alumina (C/Al₂O₃). This material provides higher retentivity and comparable selectivity, but its use is still limited by how few HPLC quality types of alumina particles (e.g., particle size, surface area, and pore size) are available. In this work, we have developed useful carbon phases on silica particles, which are available in various particle sizes, pore sizes and forms of HPLC grade. To make the carbon phase on silica, we first treat the silica surface with a monolayer or less of metal cations that bind to deprotonated silanols to provide catalytic sites for carbon deposition. After Al (III) treatment, a carbon phase is formed on the silica surface by chemical vapor deposition at 700 °C using hexane as the carbon source. The amount of Al (III) on the surface was varied to assess its effect on carbon deposition, and the carbon loading was varied at different Al (III) levels to assess its effect on the chromatographic properties of the various carbon adsorbents. We observed that use of a concentration of Al (III) corresponding to a full monolayer leads to the most uniform carbon coating. A carbon coating sufficient to cover all the Al (III) sites, required about 4–5 monolayers in this work, provided the best chromatographic performance. The resulting carbon phases behave as reversed phases with reasonable efficiency (50,000–79,000 plates/m) for non-aromatic test species.     

Liquid chromatography of macromolecules at the critical adsorption point. II. Role of column packing: Bare silica gel

Liquid chromatography of macromolecules at the critical adsorption point (LC CAP) presents a potentially very powerful method for molecular characterization of complex polymers. However, LC CAP applicability is limited due to various experimental problems. The pore sizes and surface chemistry of the column packings belong to the most important weak points of the method. The LC CAP behavior of poly(methyl methacrylate)s was investigated using bare silica gels of 6, 12, and 100 nm pore sizes and with various amounts of surface silanols. Tetrahydrofuran as the adsorption suppressing liquid and toluene as the adsorption promoting liquid were mixed to form the “nearly critical” eluents. Both pore size and surface chemistry of silica were found to strongly influence the retentive characteristics of the system in the critical adsorption area. Macromolecules that were large enough to be excluded from the packing pores hardly followed the LC CAP rules: their retention volumes changed irregularly with the polymer molar mass and their recovery dropped sharply. The narrow pore silica gel-packed column governed the elution patterns of the whole column set composed of silica gels with different pore sizes. This makes the conventional LC CAP characterization of common polymers with broader molar mass distribution impractical and even not feasible. A hybrid column system was proposed containing narrow pore nonadsorptive column added in series to the meso- and macroporous LC CAP silica gels. This narrow pore column would allow separation of gas, impurities, and system peaks from the polymer peaks. The possible successive changes of the surface of silica gel, e.g., due to formation of silanols by hydrolysis or due to irreversible adsorption of some admixtures from the sample or eluent may make the LC CAP irrepeatable. Pronounced peak broadening was observed in the critical adsorption area and this effect increased strongly with the polymer molar mass. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 1363–1371, 1998     

A reversed phase high-performance liquid chromatography method for determination of cephalexin in human plasma

A reversed-phase HPLC method for the analysis of cephalexin (7-[(aminophenylacetyl)amino]-3-methyl-8-oxo-5-thia-1-azabicyclo[4.2.0]oct-2-ene-2-carboxylic acid) by isocratic separation is described. A comparison is made between the ultrafiltration land the extraction procedure developed in our laboratory. The extraction procedure, based on the deproteinization of plasma with perchloric acid followed by the extraction with dichloroethane and separation of cephalexin on a reversed phase column, gave better recovery. Quantitative validation of our method has been performed by an external standard technique. The relative standard deviations were between 1.1 and 1.9% in the within-day assay and between 1.2 and 2.2% in the inter-day assay. The limit of detection was 0.14ug/ml and the limit of quantification was 0.28ug/ml. This method is rapid, sensitive, simple, and reproducible. The LC MS/MS technique was successfully used for identification and quantification of cephalexin in human plasma samples.     

模板法制备大孔硅胶微球及其在高效液相色谱蛋白质分离中的应用

以弱阳离子交换聚合物微球(WCX)为模板、N-三甲氧基硅基丙基-N,N,N-三甲基氯化铵(TMSPTMA)为结构导向剂、四乙氧基硅烷(TEOS)为硅胶前驱体,在三乙醇胺弱碱催化作用下,水解缩合形成有机聚合物与二氧化硅复合微球,将此复合微球煅烧后得到大孔二氧化硅微球。探索了不同反应条件对二氧化硅微球的形貌、表面结构和分散性的影响;当TMSPTMA、TEOS与三乙醇胺的体积比为1∶2∶2时可以得到孔径在50~150 nm之间、粒径在2μm左右的硅胶微球。对所制备的大孔硅胶微球表面进行C18(十八烷基二甲基氯硅烷)键合修饰,然后将键合的填料装填到50 mm×4.6 mm的色谱柱中,考察了其对常见的几种标准蛋白质和市售大豆分离蛋白质的分离效果,结果显示这种填料在高效液相色谱蛋白质分离中具有一定的潜力。     

Analytical method development for directed enzyme evolution research: A high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality l-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C₁₈ guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4 × 96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM β-cyclodextrin was used as electrolyte. 0.35%of d-ribose in l-ribose can be detected which can be translated into 99.3% ee of l-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.     

Simple method for the assay of linezolid in Brain Heart Infusion broth by high-performance liquid chromatography

A simple HPLC method was developed and validated for quantification of linezolid in Brain Heart Infusion (BHI) broth. Eperezolid was employed as internal standard and the sample pre-treatment procedure was simple. Calibration standards ranged from 0.05 to 16 mg/L. Accuracy was within 7.8% and reproducibility (RSD) was less than 8.3%. Recovery was approximately 100% at all concentrations examined. Linezolid was stable in the autosampler insert for at least 24 h at ambient temperature and in BHI for 72 h at 37 degrees C. This assay is rapid and ideal for analysis of a large number of samples.     

Simple method for the assay of clindamycin in human plasma by reversed-phase high-performance liquid chromatography with UV detector

A rapid and simple high-performance liquid chromatography (HPLC) method was developed and validated for the quantification of clindamycin in human plasma. After precipitation with 50% trichloroacetic acid (TCA) containing the internal standard, propranolol, the analysis of the clindamycin level in the plasma samples was carried out using a reverse-phase cyano (CN) column with ultraviolet detection (204 nm). The chromatographic separation was accomplished with an isocratic mobile phase consisting of acetonitrile-distilled water-7.6 mm tetramethylammonium chloride (TMA) (60:40:0.075, v/v/v), adjusted to pH 3.2. The proposed method was specific and sensitive with a lower limit of quantitation (LLOQ) of 0.2 microg/mL. This HPLC method was validated by examining the precision and accuracy for inter- and intraday analysis in the concentration range 0.2-20.0 microg/mL. The relative standard deviations (RSD) in the inter- and intraday validation were 6.1-14.9 and 6.0-16.1%, respectively. In the stability test, clindamycin was found to be stable in human plasma during the storage and assay procedure. The present HPLC method was applied to the analysis of samples taken up to 12 h after a single oral administration of clindamycin in healthy volunteers.