Ultra high performance liquid chromatography method development for separation of omeprazole and related substances on core‐shell columns using a Quality by Design approach

An updated and improved method for analysis of omeprazole/esomeprazole and related substances on core‐shell columns was developed using Fusion LC Method Development?. The method was optimized with respect to column type, column temperature, mobile phase pH level, and gradient time. Four different core‐shell columns were examined to develop a method suitable for both high performance‐ and ultra‐high performance liquid chromatography using a Quality by Design approach. The final method offers two alternative columns: Poroshell EC C18 (3.0 × 100 mm, 2.7 µm) or Poroshell HPH (3.0 × 100 mm, 2.7 µm) with the same gradient elution condition and mobile phase composition. Total run time is 18 min with 12 min of gradient elution. Phosphate buffer (15 mM, pH 7.8) is selected as the aqueous mobile phase and acetonitrile as the organic mobile phase. Column temperature is set at 40°C and ultraviolet detection at 302 nm. Furthermore, by studying parameters in a systematic way, an understanding of the effect of the input parameters enhances the method robustness and should allow for regulatory flexibility in terms of post‐approval changes. Compared to the current United States Pharmacopeia method, the updated method is faster, more efficient and performs well above acceptance criteria.     

Quality evaluation of Semen Cassiae (Cassia obtusifolia L.) by using ultra-high performance liquid chromatography coupled with mass spectrometry

A sensitive and reliable ultra-high performance liquid chromatography-electrospray ionization-tandem mass spectrometry has been developed and partially validated to evaluate the quality of Semen Cassiae (Cassia obtusifolia L.) through simultaneous determination of 11 anthraquinones and two naphtha-γ-pyrone compounds. The analysis was achieved on a Poroshell 120 EC-C(18) column (100 mm × 2.1 mm, 2.7 μm; Agilent, Palo Alto, CA, USA) with gradient elution using a mobile phase that consisted of acetonitrile-water (30 mM ammonium acetate) at a flow rate of 0.4 mL/min. For quantitative analysis, all calibration curves showed perfect linear regression (r(2) > 0.99) within the testing range. This method was also validated with respect to precision and accuracy, and was successfully applied to quantify the 13 components in nine batches of Semen Cassiae samples from different areas. The performance of developed method was compared with that of conventional high-performance liquid chromatography method. The significant advantages of the former include high-speed chromatographic separation, four times faster than high-performance liquid chromatography with conventional columns, and great enhancement in sensitivity. This developed method provided a new basis for overall assessment on quality of Semen Cassiae.     

Determination of Saponins in Leaf of Panax Ginseng C. A. Mey. by High Performance Liquid Chromatography

A high performance liquid chromatography(HPLC) with UV detection was established for simultaneous determination of saponins in the leaf of Panax ginseng C. A. Mey. Nine ginsenosides(Rb1,Rb2,Rb3,Rc,Rd,F1,F2,F3,F5) and notoginsenoside Fe(NFe) were studied. Among the saponins,the ginsenosides F1,F2,F3,F5 and NFe were determined by HPLC-UV method for the first time. The determination of the ginsenosides via the HPLC-UV method was performed on a reversed-phase C18 column with gradient elution in 40 min. The line...     

Deformation and degradation of polymers in ultra-high-pressure liquid chromatography

Ultra-high-pressure liquid chromatography (UHPLC) using columns packed with sub-2 μm particles has great potential for separations of many types of complex samples, including polymers. However, the application of UHPLC for the analysis of polymers meets some fundamental obstacles. Small particles and narrow bore tubing in combination with high pressures generate significant shear and extensional forces in UHPLC systems, which may affect polymer chains. At high stress conditions flexible macromolecules may become extended and eventually the chemical bonds in the molecules can break. Deformation and degradation of macromolecules will affect the peak retention and the peak shape in the chromatogram, which may cause errors in the obtained results (e.g. the calculated molecular-weight distributions). In the present work we explored the limitations of UHPLC for the analysis of polymers. Degradation and deformation of macromolecules were studied by collecting and re-injecting polymer peaks and by off-line two-dimensional liquid chromatography. Polystyrene standards with molecular weight of 4 MDa and larger were found to degrade at UHPLC conditions. However, for most polymers degradation could be avoided by using low linear velocities. No degradation of 3-MDa PS (and smaller) was observed at linear velocities up to 7 mm/s. The column frits were implicated as the main sources of polymer degradation. The extent of degradation was found to depend on the type of the column and on the column history. At high flow rates degradation was observed without a column being installed. We demonstrated that polymer deformation preceded degradation. Stretched polymers eluted from the column in slalom chromatography mode (elution order opposite to that in SEC or HDC). Under certain conditions we observed co-elution of large and small PS molecules though a convolution of slalom chromatography and hydrodynamic chromatography.     

Core–shell column Tanaka characterization and additional tests using active pharmaceutical ingredients

In the last decade, core–shell particles have gained more and more attention in fast liquid chromatography separations due to their comparable performance with fully porous sub‐2 μm particles and their significantly lower back pressure. Core–shell particles are made of a solid core surrounded by a shell of classic fully porous material. To embrace the developed core–shell column market and use these columns in pharmaceutical analytical applications, 17 core–shell C₁₈ columns purchased from various vendors with various dimensions (50 mm × 2.1 mm to 100 mm × 3 mm) and particle sizes (1.6–2.7 μm) were characterized using Tanaka test protocols. Furthermore, four selected active pharmaceutical ingredients were chosen as test probes to investigate the batch to batch reproducibility for core–shell columns of particle size 2.6–2.7 μm, with dimension of 100 × 3 mm and columns of particle size 1.6 μm, with dimension 100 × 2.1 mm under isocratic elution. Columns of particle size 2.6–2.7 μm were also tested under gradient elution conditions. To confirm the claimed comparable efficiency of 2.6 μm core–shell particles as sub‐2 μm fully porous particles, column performances of the selected core–shell columns were compared with BEH C₁₈, 1.7 μm, a fully porous column material as well.     

A study of the effects of column porosity on gradient separations of proteins

The type of the stationary phase for reversed-phase liquid chromatography significantly affects the sample elution. Hydrodynamic properties, efficiency and gradient elution of proteins were investigated on five commercial C18 columns with wide-pore totally porous particles, with superficially porous layer particles, non-porous particles and a silica-based monolithic bed. The efficiency in the terms of reduced plate height is higher for low-molecular ethylbenzene than for proteins, but depends on the character of the pores in the individual columns tested. The superficially porous Poroshell and the non-porous Micra columns provide the best efficiency for proteins at high mobile phase flow rates, probably because of similar pore architecture in the stationary phase. The Zorbax column with similar pore architecture as the Poroshell active layer, i.e. narrow pore distribution of wider pores shows better efficiency than the packed column with narrow pores and broad pore distribution. The monolithic column shows lower efficiency for proteins at high flow rates, but it performs better than the broad-pore distribution totally porous particulate columns. Different pore architecture affects also the retention and selectivity for proteins on the individual columns. The retention times on all columns can be predicted using the model for reversed-phase gradient elution developed originally for low-molecular compounds. Consideration of the limited pore volume accessible to the biopolymers has negligible effect on the prediction of retention on the columns packed with non-porous or superficially porous particles, but improves the accuracy of the predicted data for the totally porous columns with broad pore distribution.     

Separation of cannabinoids on three different mixed‐mode columns containing carbon/nanodiamond/amine‐polymer superficially porous particles

Three mixed‐mode high‐performance liquid chromatography columns packed with superficially porous carbon/nanodiamond/amine‐polymer particles were used to separate mixtures of cannabinoids. Columns evaluated included: (i) reversed phase (C₁₈), weak anion exchange, 4.6 × 33 mm, 3.6 μm, and 4.6 × 100 mm, 3.6 μm, (ii) reversed phase, strong anion exchange (quaternary amine), 4.6×33 mm, 3.6 μm, and (iii) hydrophilic interaction liquid chromatography, 4.6 × 150 mm, 3.6 μm. Different selectivities were achieved under various mobile phase and stationary phase conditions. Efficiencies and peak capacities were as high as 54 000 N/m and 56, respectively. The reversed phase mixed‐mode column (C₁₈) retained tetrahydrocannabinolic acid strongly under acidic conditions and weakly under basic conditions. Tetrahydrocannabinolic acid was retained strongly on the reversed phase, strong anion exchange mixed‐mode column under basic polar organic mobile phase conditions. The hydrophilic interaction liquid chromatography column retained polar cannabinoids better than the (more) neutral ones under basic conditions. A longer reversed phase (C₁₈) mixed‐mode column (4.6 × 100 mm) showed better resolution for analytes (and a contaminant) than a shorter column. Fast separations were achieved in less than 5 min and sometimes 2 min. A real world sample (bubble hash extract) was also analyzed by gradient elution.     

Analysis of global components in Ganoderma using liquid chromatography system with multiple columns and detectors

In present study, a multiple columns and detectors liquid chromatography system for analysis of global components in traditional Chinese medicines was developed. The liquid chromatography system was consist of three columns, including size exclusion chromatography column, hydrophilic interaction chromatography column, and reversed phase chromato‐graphy column, and three detectors, such as diode array detector, evaporative light scattering detector, and mass spectrometry detector, based on column switching technique. The developed multiple columns and detectors liquid chromatography system was successfully applied to the analysis of global components, including macromolecular (polysaccharides), high (nucleosides and sugars)‐, and low (triterpenes)‐polarity small molecular compounds in Ganoderma, a well‐known Chinese medicinal mushroom. As a result, one macromolecular chromatographic peak was found in two Ganoderma species, 19 components were identified in Ganoderma lucidum (two sugars, three nucleosides, and 14 triterpenes), and four components (two sugars and two nucleosides) were identified in Ganoderma sinense. The developed multiple columns and detectors liquid chromatography system was helpful to understand comprehensive chemical characters in TCMs.     

Multilayered particle‐packed column: Evaluation and comparison with monolithic and core–shell particle columns for the determination of red azo dyes in Sequential Injection Chromatography

A recently presented new type of “multilayered” organic–inorganic hybrid silica particle packed column YMC‐Triart C₁₈ (50 mm × 4.6 mm, 5 μm) was used for the development of a sequential injection chromatography method for determination of five azo dyes (Sudan I, Sudan II, Sudan III, Sudan orange G, and para red) in selected food seasonings. The use of a novel sorbent brings attractive features, reduced backpressure, and broader chemical stability together with high separation performance, which are discussed and compared with that of three types of columns typically used in medium‐pressure flow chromatography techniques (classic monolithic, narrow monolithic, and core–shell particle columns). The separation was performed in gradient elution mode created by the zone mixing of two mobile phases (acetonitrile/water 90:10, 1.5 mL + acetonitrile/water 100:0, 2.3 mL) at a flow rate of 0.60 mL/min and time of analysis <9.5 min. The spectrophotometric detection wavelengths were set to 400, 480, and 500 nm. The high performance of the developed method with multilayered particle column was well documented and the results indicate a broad capability of sequential injection chromatography.     

Molybdenum Speciation in Raw Phloem Sap of Castor Bean

Abstract

Separation and quantification of molybdenum (Mo) in raw phloem sap from castor bean (Ricinus communis L.) was performed by size exclusion chromatography (SEC) and further purification was performed using quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE). For elemental detection, an inductively coupled plasma quadrupole mass spectrometer (ICP-QMS) was applied. Two different SEC columns were utilized: column A, Sephadex G-50 SF (700 mm × 24 mm), and column B, Sephadex G-25 M (28 mm × 9 mm). The protein content of the fractions was determined by the Bradford method. Using column A, two peaks of Mo were detected consisting of a main peak (Mo_A2) in the low molecular weight area (< 1.35 kDa), and a minor peak (Mo_A1, ≥ 30 kDa) at the void volume of the column. Both Mo species were detected at the ultraviolet (UV) active absorption area of raw phloem sap. Two peaks of Mo were also detected using column B, the first peak (Mo_B1) being at the same elution volume as the protein of raw phloem sap, and the second one (Mo_B2) was eluted in the area of 1.5 to 2.4 mL of elution volume. Raw phloem sap digested by proteinase K-enzyme indicates a significant reduction of Mo_B1 peak, which suggests that the peak may contain Mo bound to protein or polypeptides. The raw phloem sap and SEC fraction containing highest Mo concentration (Mo_A2) were furthermore separated by QPNC-PAGE. The result reveals that the Mo-containing fraction from the raw phloem sap was eluted at the same retention volume as the purified sample. This implies that the Mo species were also successfully separated and purified using QPNC-PAGE.     

Purification of saponins from leaves of Panax notoginseng using preparative two-dimensional reversed-phase liquid chromatography/hydrophilic interaction chromatography

Saponins are widely distributed in the plant kingdom and have been shown to be active components of many medicinal herbs. In this study, a two-dimensional purification method based on reversed-phase liquid chromatography coupled with hydrophilic interaction liquid chromatography was successfully applied to purify saponins from leaves of Panax notoginseng. Nine saponin reference standards were used to test the separation modes and columns. The standards could not be resolved using C₁₈ columns owing to their limited polar selectivity. However, they were completely separated on a XAmide column in hydrophilic interaction liquid chromatography mode, including two pairs of standards that were coeluted on a C₁₈ column. The elution order of the standards on the two columns was sufficiently different, with a correlation coefficient between retention times on the C₁₈ and XAmide columns of 0.0126, indicating good column orthogonality. Therefore, the first-dimension preparation was performed on a C₁₈ column, followed by a XAmide column that was used to separate the fractions in the second dimension. Fifty-four fractions were prepared in the first dimension, with 25 fractions rich in saponins. Eight saponins, including two pairs of isomeric saponins and one new saponin, were isolated and identified from three representative fractions. This procedure was shown to be an effective approach for the preparative isolation and purification of saponins from leaves of P. notoginseng. Moreover, this method could possibly be employed in the purification of low-content and novel active saponins from natural products.

硅胶基硝化苯硼酸亲和色谱材料的合成及应用

合成了硅胶基硝化苯硼酸亲和色谱材料,首先对间氨基苯硼酸进行硝基化,制得3-氨基-4-硝基苯硼酸功能基团,通过γ-缩水甘油醚氧丙基三甲氧基硅烷把功能基团键合到硅胶基体上,在20.7MPa压力下装成亲和色谱预柱(35mm×4.6mmi.d.)。用该预柱通过六通阀后接ODS分析柱(250mm×4.6mmi.d.),构成中心切割二维HPLC。该系统能对含有顺二羟基结构的化合物进行在线富集,实现生物复杂样品直接进样分离分析。使用该系统对尿中多种修饰核苷进行了分离分析,以pH值7.95的0.25mol/LNH4Ac碱性缓冲液把实际尿样(100μL)中核苷保留在预柱上,生物大分子无保留通过,再切换六通阀,以pH4.50的25mmol/LKH2PO4酸性洗脱液把保留在预柱上的核苷洗脱,进样到下一级ODS分析柱柱头上聚焦,然后用梯度洗脱法(pH4.50的25mmol/LKH2PO4缓冲液与体积比60∶40的甲醇-水梯度混合构成洗脱液)完成核苷在ODS分析柱上的分离(紫外检测260nm),11种目标核苷的分离分析取得了良好的定性定量结果。     

Practical comparison of LC columns packed with different superficially porous particles for the separation of small molecules and medium size natural products

Commercial C(18) columns packed with superficially porous particles of different sizes and shell thicknesses (Ascentis Express, Kinetex, and Poroshell 120) or sub-2-μm totally porous particles (Acquity BEH) were systematically compared using a small molecule mixture and a complex natural product mixture as text probes. Significant efficiency loss was observed on 2.1-mm id columns even with a low dispersion ultra-high pressure liquid chromatography system. The Kinetex 4.6-mm id column packed with 2.6-μm particles exhibited the best overall efficiency for small molecule separations and the Poroshell 120 column showed better performance for mid-size natural product analytes. The Kinetex 2.1-mm id column packed with 1.7-μm particles did not deliver the expected performance and the possible reasons besides extra column effect have been proved to be frictional heating effect and poor column packing quality. Different column retentivities and selectivities have been observed on the four C(18) columns of different brands for the natural product separation. Column batch-to-batch variability that has been previously observed on the Ascentis Express column was also observed on the Kinetex and Poroshell 120 column.     

Improved Extraction Method and Chromatographic Separation of Ginsenosides from Ginseng Roots,Leaves and Ginseng Drug Preparations

A new extraction method for ginsenosides from ginseng roots, ginseng leaves and ginseng drug preparations by Sep-Pak C₁₈ cartridges has been studied. Ginsenoside extraction by Sep-Pak cartridges is a rapid, efficient, reproducible method. In addition, the extracts were analyzed by high performance thin layer chromatography (HPTLC) and reverse phase high performance liquid chromatography (HPLC). The major components of ginseng saponins were effectively separated using an ODS-120T column.     

Studies on high-performance size-exclusion chromatography of synthetic polymers. I. Volume of silica gel column packing pores reduced by retained macromolecules

Macromolecules, which stay adsorbed within the active size-exclusion chromatography (SEC) column packings may strongly reduce effective volume of the separation pores. This brings about a decrease of retention volumes of the non-retained polymer samples and results in the increased apparent molar mass values. The phenomenon has been demonstrated with a series of poly(methyl methacrylate)s (PMMA) and a polyethylenoxide (PEO) fully retained by adsorption within macroporous silica gel SEC column from toluene or tetrahydrofuran, respectively. The non-retained probes were polystyrenes (PS) in toluene and both PS and PMMA in THF eluents. The errors in the peak molar mass values determined for the non-retained polymer species using a column saturated with adsorbed macromolecules and considering calibration curves monitored for the original "bare" column packing assumed up to several hundreds of percent. Errors may appear also in the weight and number averages of molar masses calculated from calibration dependences obtained with columns saturated with adsorbed macromolecules. Moreover, the SEC peaks of species eluted from the polymer saturated columns were broadened and in some cases even split. These results demonstrate a necessity not only to periodically re-calibrate the SEC columns but also to remove macromolecules adsorbed within packing in the course of analyses.     

Meropenem and ciprofloxacin in complicated gastric surgery for cancer patients: A simple SPE–UHPLC–PDA method for their determination in human plasma

A simple and rapid ultra‐high‐performance liquid chromatographic (UHPLC) for the simultaneous determination of meropenem and ciprofloxacin in human plasma was developed and validated. All of the analytes were separated in <5 min. A solid‐phase extraction method was applied from sample preparation. Analytical separation was performed on a Poroshell SB C₁₈ column (50 × 2.1 mm, 2.7 μm particle size) with photodiode array (PDA) detection. Meropenem and ciprofloxacin were determined at wavelengths of 300 and 277 nm, respectively. The mobile phase was a mixture of acetonitrile–10 mm ammonium acetate–methanol in gradient elution. The method has been validated for both drugs in gastric surgery for cancer patients. The method showed good linearity with correlation coefficients, r² = 0.994 for the two drugs, as well as high precision (RSD < 10.5% in each case); accuracy ranged from ?5.8 to +6.0%. The limit of quantitation of the two drugs was established at 0.02 and 0.01 μg/mL, respectively. Meropenem, ciprofloxacin and the internal standard were extracted from human plasma with a mean recovery ranging from 92.5 to 98.6%. The method was applied to quantify the drugs dosage in complicated gastric surgery patients.     

Determination of chikusetsusaponin V and chikusetsusaponin IV in rat plasma by liquid chromatography–mass spectrometry and its application to a preliminary pharmacokinetic study

A sensitive liquid chromatography–electrospray ionization–mass spectrometry method has been developed and validated for determination of two major bioactive saponins in rat plasma after oral administration of saponins extracted from Rhizoma Panacis Japonici, including chikusetsusaponin V and chikusetsusaponin IV for the first time. Akebia saponin D was used as the internal standard (IS). Plasma samples were prepared by protein precipitation with methanol. A Phenomenex C₁₈ column (150 × 4.6 mm, 4 µm) was used as the analytical column with a mobile phase of acetonitrile and 0.05% aqueous formic acid. Mass spectrometric detection was achieved by single quadrupole mass spectrometer equipped with an electrospray ionization interface operating in negative ionization mode. Calibration curves showed good linearity over the concentration range of 5–500 ng/mL for the two analytes in rat plasma. The lower limit of quantification was 5 ng/mL. The intra‐ and inter‐batch precisions were within 10.3% and accuracy ranged from ?3.9 to 5.4%. The method was validated and successfully applied to the preliminary pharmacokinetic study of chikusetsusaponin V and chikusetsusaponin IV in rat plasma after oral administration of saponins extracted from Rhizoma Panacis Japonici. Copyright © 2013 John Wiley & Sons, Ltd.     

Efficient method for large-scale preparation of two components H and I of Sg-6 saponins from whole seeds of wild soybean (Glycine soja Sieb. and Zucc.)

New saponin components, Sg-6 saponins, have been recently reported from the seeds of wild soybean (Glycine soja) which may have specific health benefits. To evaluate the possible health benefits, a large amount of Sg-6 saponins are needed, but general group A acetyl saponins and new Sg-6 saponins are eluted in overlapping peaks by ordinal preparative high-performance liquid chromatography and/or open column methods. A new method is proposed in this report. This method includes (1) deacetylation of group A acetyl saponins in alkali condition with KOH, (2) precipitation of Sg-6 saponins in acid condition with HCl, (3) recovery of Sg-6 saponins with aqueous methanol from the precipitate, and (4) elution of Sg-6 saponins by preparative reverse-phase open column. With this method, from 450?g of wild soybean whole seed powder, about 1?g of Sg-6 saponins (mixture of six components) was clearly separated from other saponins with 61% recovery.     

High-resolution monolithic columns—a new tool for effective and quick separation

This work describes a comparison of three types of commercial high-performance liquid chromatography silica monolithic columns with different inner diameters and generations of monolithic sorbent: a “classic” monolithic column, the first generation (Onyx? monolithic C₁₈, 100 mm?×?4.6 mm, Phenomenex); a “narrow” monolithic column for fast separation at lower flow rates (Chromolith® Performance RP-18e, 100 mm?×?3 mm, Merck); and a recently introduced “high-resolution” monolithic column, the next generation (Chromolith® HighResolution RP-18e, 100 mm?×?4.6 mm, Merck). Separation efficiency (number of theoretical plates, height equivalent to a theoretical plate and van Deemter curves), working pressure, the symmetry factor and resolution were critical aspects of the comparison in the case of the separation of ascorbic acid, paracetamol and caffeine. The separations were performed under isocratic conditions with a mobile phase consisting of 10:90 (v/v) acetonitrile–phosphoric acid (pH 2.80). Detailed comparison of the newest-generation monolithic column (Chromolith® HighResolution) with the previously introduced monolithic sorbents was performed and proved the advantages of the Chromolith® HighResolution column.