Estimation of Enantiomeric Impurity in Piperidin‐3‐Amine by Chiral HPLC With Precolumn Derivatization

A simple, precise, accurate, robust chiral high‐performance liquid chromatographic (chiral HPLC) method was developed for estimation of (S)‐piperidin‐3‐amine (S‐isomer) in (R)‐piperidin‐3‐amine dihydrochloride (R‐AMP). As AMP is a high‐melting solid and nonchromophoric compound, development of a suitable chiral method is a challenging task. The proposed chiral HPLC‐UV method involves a precolumn derivatization technique with para toluene sulphonyl chloride (PTSC) in the presence of a base to introduce chromophore into analytes. It utilizes chiralpak AD‐H column with a simple mobile phase of 0.1% diethyl amine in ethanol with a 0.5 mL/min flow rate. Analytes were monitored by using a UV detector at 228 nm. The resolution between the two enantiomers was more than 4.0. The developed method was validated as per current International Conference on Harmonization (ICH) guidelines. Chirality 26:775–779, 2014. © 2014 Wiley Periodicals, Inc.     

A method for the quantitative enantioselective HPLC analysis of ethambutol and its stereoisomers

A rapid, quantitative, enantioselective HPLC method, using N,N′-dibenzylethylenediamine as internal standard, for the analysis of the three stereoisomers of the antituberculosis drug ethambutol and 2-aminobutan-1-ol is described. The derivatisation and chromatographic procedures and their reproducibility, durability, and sensitivity are discussed. Application to assays of raw material, tablet content, and synthetic reactions are illustrated. The method involves perbenzoylation, in pyridine, followed by direct HPLC on a “Pirkle covalent bonded column” containing a 3,5-dinitrobenzoyl-D -phenylglycine as chiral stationary phase (CSP) using hexane/IPA as mobile phase and detection at 230 nm.     

Estimation of scopoletin from a polyherbal formulation composition using HPLC coupled with fluorescence detector through the concept of Design of Experiment

High-performance liquid chromatography (HPLC) coupled with a fluorescence detector was used to analyse bioactive phytoconstituent scopoletin from a polyherbal composition derived from the extract prepared from roots of Argyreia nervosa, roots of Withania somnifera, and fruits of Tribulus terrestris. This analytical method was developed as a quality control tool for standardization of the composition to be formulated to enhance spermatogenesis. Chromatographic separation was achieved using Luna® (250 mm × 4.6 mm, 100 Å, 5 μm) C₁₈ column as a stationary phase, and water (0.01 M glacial acetic acid):methanol: acetonitrile (60:20:20, %v/v/v) as the mobile phase; passed through the column at a set flow rate of 1.0 ml min⁻¹. The elute in the flow cell was excited at 345 nm and the chromatogram was recorded at 444 nm as the emission wavelength. As a part of the analytical Quality by Design approach, systemic studies were conducted to identify potential risks affecting the critical attributes (area, resolution, retention time) of the analytical method, and mitigating the potential risks after optimizing the chromatographic parameters with the help of the Design of Experiment approach. The developed analytical method was subjected to the validation studies, which showed a linear relationship (r² = 0.9982) between the concentration and the area corresponding to scopoletin peak in the concentration range 10–130 ng ml⁻¹. The method was found selective, sensitive, and precise. The recovery of the scopoletin was found in a range 99.53–102.13%; confirming the accuracy of the analytical method. The amount of scopoletin was estimated to be 0.146%w/w from the polyherbal composition.     

Manual and automatic extraction and high-performance liquid chromatographic determination of a spicamycin derivative, KRN5500, in rat plasma

A sensitive reliable method for the extraction, separation and quantitation of KRN5500 (I), a spicamycin derivative, from rat plasma was developed. It involves solid-phase extraction of the drug using a Bond Elut C₁₈ cartridge and reversed-phase HPLC on a YMC-Pack ODS column with an ultraviolet detector. The intra- and inter-assay coefficients of variation by manual (n=10) and automatic (n=5) extraction were less than 9 and 13% and 6 and 8%, respectively. The limit of quantitation of each extraction procedure was 2 ng potency/ml. This extraction method may thus be considered useful for monitoring I in animals following its administration.     

Reliable HPLC separation,vibrational circular dichroism spectra,and absolute configurations of isoborneol enantiomers

Resolution of chiral compounds has played an important role in the pharmaceutical field, involving detailed studies of pharmacokinetics, physiological, toxicological, and metabolic activities of enantiomers. Herein, a reliable method by high‐performance liquid chromatography (HPLC) coupled with an optical rotation detector was developed to separate isoborneol enantiomers. A cellulose tris(3, 5‐dimethylphenylcarbamate)‐coated chiral stationary phase showed the best separation performance for isoborneol enantiomers in the normal phase among four polysaccharide chiral packings. The effects of alcoholic modifiers and column temperature were studied in detail. Resolution of the isoborneol racemate displayed a downward trend along with an increase in the content of ethanol and column temperature, indicating that less ethanol in the mobile phase and lower temperature were favorable to this process. Moreover, two isoborneol enantiomers were obtained via a semipreparative chiral HPLC technique under optimum conditions, and further characterized by analytical HPLC, and experimental and calculated vibrational circular dichroism (VCD) spectroscopy, respectively. The solution VCD spectrum of the first‐eluted component was consistent with the Density Functional Theory (DFT) calculated pattern based on the SSS configuration, indicating that this enantiomer should be (1S , 2S , 4S )‐(+)‐isoborneol. Briefly, these results have provided reliable information to establish a method for analysis, preparative separation, and absolute configuration of chiral compounds without typical chromophoric groups.     

Simple Isocratic HPLC Method for Determination of Enantiomeric Impurity in Besifloxacin Hydrochloride

Besifloxacin is a unique chiral broad‐spectrum flouroquinolone used in the treatment of bacterial conjunctivitis. R‐form of besifloxacin hydrochloride shows higher antibacterial activity as compared to the S‐isomer. Therefore, it is necessary to establish chiral purity. To establish chiral purity a high‐performance liquid chromatography (HPLC) method for determination of R‐besifloxacin and S‐besifloxacin (BES impurity A) was developed and validated for in‐process quality control and stability studies. The analytical performance parameters such as linearity, precision, accuracy, specificity, limit of detection (LOD), and lower limit of quantification (LOQ) were determined according to International Council for Harmonization ICH Q2(R1) guidelines. HPLC separation was achieved on Chiralpak AD‐H (250 x 4.6 mm, 5 μm) column using n‐heptane: ethanol: ethylenediamine: acetic acid (800:200:0.5:0.5) (v/v/v/v) as the mobile phase in an isocratic elution. The eluents were monitored by UV/Visible detector at 290 nm. The resolution between S‐isomer and besifloxacin hydrochloride was more than 2.0. Based on a signal‐to‐noise ratio of 3 and 10 the LOD of besifloxacin was 0.30 μg/mL, while the LOQ was 0.90 μg/mL. The calibration curves were linear in the range of 0.9–7.5 μg/mL. Precision of the method was established within the acceptable range. The method was suitable for the quality control enantiomeric impurity in besifloxacin hydrochloride. Chirality 28:628–632, 2016. © 2016 Wiley Periodicals, Inc.     

Optimization of an HPLC peroxyoxalate chemiluminescence detection system for some dansyl amino acids

Bis(2,4,6-trichlorophenyl) oxalate (TCPO)-hydrogen-peroxide-generated chemiluminescence (CL) of four dansyl amino acids has been used as a model system for the optimization of a detection system in reversed-phase high-performance liquid chromatography. Dansylated alanine, glutamic acid, methionine, and norleucine were subjected to peroxyoxalate induced CL in a static system and in a flow system under various conditions with respect to TCPO (ethyl acetate) and hydrogen peroxide (acetone) concentrations, solvent composition and flow, using a two-pump or a one-pump post-column reagent system. From the CL-decay curve, the influence on the emission signal from the total flow rate in the detector was investigated. Special attention was focused on the mixing of the LC eluate and the reagent in order to combine an efficient collection of the emitted light using a 74μI flow cell (originally 10μI in the fluorescence detector) with minimal extra column band broadening. Therefore, a capillary fused-silica tubing of about 100μm i.d. was inserted against the end-frit of the column and brought through a mixing tee, in which the solutions of TCPO and hydrogen peroxide were added. The column end tubing ended in the flow cell and the LC eluate and the reagents were mixed when entering the flow-cell. Average detection limits (S/N=2) of 200fmol injected dansylated amino acid could be reached. A comparison is made between the use of TCPO and DNPO (bis (2, 4-dinitrophenyl) oxalate).     

Coupled chromatography on Sepharose 4B-cellulose nitrate columns for the isolation of an R-plasmid fromPseudomonas aeruginosa

A chromatographic technique for isolating bacterial plasmid DNA using a Sepharose 4B column connected to a cellulose nitrate column is described. After loading the Sepharose 4B column with the alkali-treated cleared bacterial lysate, the plasmid DNA was eluted from the cellulose nitrate column by passing 2 M NaCl-0.01 M sodium citrate buffer through the connected columns. This one-step procedure allows separation of plasmid DNA at a concentration suitable for direct electron microscopy. This method was applied to the separation and electron microscopic examination of a newPseudomonas aeruginosa plasmid (pYMB1).     

用高效液相色谱法(HPLC)测定肌苷中有关物质和降解产物

用HPLC测定肌苷中有关物质和降解产物 ,与主药有良好的分离效果。色谱柱为HypersilC1 8(2 5 0mm× 4 .6mm ,5 μm) ,流动相甲醇 -水 (甲醇与水体积比为 10∶90 ) ,流速 1mL·min- 1 ,检测波长 2 4 8nm。     

Simultaneous determination of byak-angelicin and oxypeucedanin hydrate in rat plasma by column-switching high-performance liquid chromatography with ultraviolet detection

A simple and sensitive column-switching HPLC method was developed for the simultaneous determination of two furocoumarin compounds, byak-angelicin and oxypeucedanin hydrate, which are the main components of hot water extract of Angelica dahurica root (AE), in rat plasma. Plasma sample was simply deproteinated with perchloric acid. After centrifugation, the supernatant was injected into a column-switching HPLC system consisting of a clean-up column (Symmetry Shield RP 8, 20×3.9 mm I.D.) and analytical column (Symmetry C₁₈, 75×4.6 mm I.D.) which were connected with a six-port switching valve. The flow-rate of the mobile phase (acetonitrile–water, 20:80) was maintained at 1 ml/min. Detection was carried out at wavelength 260 nm with a UV detector. The column temperature was maintained at 40°C. The calibration curves of byak-angelicin and oxypeucedanin hydrate were linear over the ranges 19.6 to 980 ng/ml (r²>0.997). The accuracy of these analytes was less than 4.4%. The intra- and inter-day relative standard deviations of byak-angelicin and oxypeucedanin hydrate were within 12.0% and 12.7%, respectively. The present method was applied for the analysis of plasma concentration from rats after administration of AE.     

乙醇系统HPLC分析PTC－氨基酸的条件优化

首次报道用乙醇系统分析ＰＴＣ－氨基酸的新方法中各衍生物获得最佳分离的建立过程。ＰＴＣ－氨基酸衍生后溶于Ａ溶液,然后进样于４μｍＮｏｖａＰａｋＣ１８柱（３．９ｍｍ×１５０ｍｍ）。系统的优化步序包括全面调控流动相的ｐＨ值与ＴＥＡ浓度、乙醇梯度程序、柱温等诸多影响ＨＰＬＣ色谱行为的因素。最适条件为：Ａ溶液含０．１４ＭＧ酸钠、０．７５ｍｌ／ＬＴＥＡ、ＰＨ６．３５;Ｂ溶液为１００％乙醇;柱温３０℃。通过优化的乙醇梯度最终在约４４ｍｉｎ内将１５种ＰＴＣ－氨基酸很好地分离。此法可用于替代代表新科技水平的ＰＴＣ－氨基酸乙腈分析系统。     

Stereospecific HPLC method for the quantitation of the enantiomers of MK-0571, a potent leukotriene D4 receptor antagonist, in biological specimens.

A stereospecific HPLC bioanalytical method was developed for quantitation of the enantiomers of MK-0571, a leukotriene D4 receptor antagonist. The procedure involves the addition of an internal standard analog to the biological matrix followed by extraction of the free acids into ethyl acetate. The acids are subsequently reacted with the homochiral reagent, (+)-(R)-alpha-(1-naphthyl)ethylamine (NEA) to form diastereomers. Following removal of excess reagent and side products by a dilute acid wash, the NEA-MK-0571 diastereomers are separated on a phenyl urea chiral column using a mobile phase containing hexane, isopropanol, and acetonitrile and are detected with a fluorescence detector. The sensitivity of the method is such that 50 ng of each enantiomer can be quantitated. In the 0.05 to 10 micrograms range the recoveries of the enantiomers of MK-0571 from plasma were 100.4 +/- 7.9% and 100.0 +/- 7.2%. NMR and mass spectral data confirmed the structure of the derivative. The method has been utilized in drug safety evaluation studies to demonstrate enantioselectivity in disposition of the enantiomers of MK-0571 in rats and monkeys but not in mice.     

Characterization of biotechnological processes and products using high-performance liquid chromatography (H P L C). VII. Determination of organic compounds in sulphite liquor

Carbohydrates, acetic acid, furfural, 5-hydroxymethylfurfural (HMF) were analyzed in sulphite liquor using HPLC with refractive index detection. Pentoses (xylose, arabinose) and hexoses (glucose, galactose, mannose) were separated on an Aminex HPX-87P column as wel as by means of a self-made glass cartridge packed with a piperazine impregnated silica gel. Before injecting into the HPLC apparatus the sulphite liquors were purified using a presep column packed with a cation-exchange resin. The sugar quantification between the two different separation systems was satisfactory. Using an Aminex HPX-87P column and an UV monitor set at 283 nm connected in parallel with the RI detector also furfural and HMF could be analyzed in sulphite liquor. Furthermore, an Aminex HPX-87H column and an UV detector set at 210 nm were most suitable to quantify ethanol. These modified and optimized liquid chromatographic separation systems are the basis for further investigations of the utilization of hexoses as well as pentoses in sulphite liquors by means of suitable microorganisms.     

HPLC法测定小叶山葡萄叶中白藜芦醇含量

建立一种高效液相色谱法(HPLC)测定小叶山葡萄Vitis thunbergii var.taiwaniana叶中白藜芦醇含量的检测方法。Waters高效液相色谱仪:Hypersil C18色谱柱(250 mm×4.6 mm,5μm);流动相:甲醇-水(72:28);流速:1 mL·min^-1;检测波长306 nm。结果表明,在上述色谱条件下,白藜芦醇含量在10~200μg·mL^-1范围内线性关系良好,相关系数r为0.9999。精密度、重现性、稳定性的RSD(n=5)分别为0.77%、0.41%、0.21%;平均加标回收率为99.92%,相对标准偏差为0.39%。该方法准确、灵敏、可靠,可用于白藜芦醇的定量和定性分析。     

毛脉酸模不同生长发育期HPLC色谱指纹图谱研究

采用HPLC-DAD方法,梯度洗脱,对10批不同生长发育期毛脉酸模根样品进行主成分分析。色谱条件为:Planetsil C18分析柱(5μm, 200 mm×4.6 mm),预柱Phenomenex ODS-C18(4×3.0 mm ID),柱温40℃,流动相A为甲醇;流动相B为水(磷酸调pH值为2.0),流动相A从30%甲醇到100%甲醇,时间为0~50 min,检测波长254 nm。10批毛脉酸模样品得到的色谱指纹图谱有27个共有峰,其特征峰指纹图谱可分为Ⅰ, Ⅱ, Ⅲ三组:第Ⅰ组包括0~17 min(1~14号峰),第Ⅱ组包括17~35 min(15~24号峰),第Ⅲ组包括35~50 min(25~27号峰)。HPLC-DAD方法分析毛脉酸模主成分,方法准确可靠,其本身具有多成分同时定性的优势;27个共有峰的出峰先后顺序及相对含量极具特征性、专属性,重复性好,形成了毛脉酸模特有的HPLC色谱指纹图谱,可作为毛脉酸模内在质量评价、鉴定及其最佳采收期确定提供科学依据。     

Trehalose analysis using ion exchange HPLC coupled with electrochemical detection

A method has been developed to detect trehalose in yeast extracts down to 10mM. A crude yeast extract was prepared by rapidly heating filtered cells to 95°C. Trehalose was separated using an ion exchange HPLC connected to an electrochemical detector.     

Development of an HPLC method for the determination of doripenem in human and mouse serum

A HPLC method utilizing solid phase extraction was developed to analyze doripenem (formerly S-4661) in human and mouse serum. A reversed-phase column was used with a UV detector set at 295 nm. The mobile phase consisted of methanol and phosphate buffer at a flow rate of 1.5 ml/min. Meropenem was used as the internal standard. The standard curve was linear over a range of 0.5-40 microg/ml. The assay is simple, reproducible, and accurate and has been used successfully to analyze doripenem concentrations from a murine pharmacokinetic study.     

HPLC法测定A群C群脑膜炎球菌多糖疫苗中乳糖含量

建立了高效液相色谱法测定A群C群脑膜炎球菌多糖疫苗中乳糖的含量。样品经离心除去多糖后,采用阳离子交换柱分离,外标法定量分析。该法线性相关系数大于0.9999,回收率为98.8%,成品测定的CV值为1.2%(<2%),该法定量准确,重复性好,适于对A群C群脑膜炎球菌多糖疫苗中乳糖含量进行快速检测。     

HPLC fluorescence method for the determination of nizatidine in human plasma and its application to pharmacokinetic study

A sensitive high‐performance liquid chromatographic (HPLC) method was developed for the determination of nizatidine in human plasma. Nizatidine was derivatized by 4‐fluoro‐7‐nitrobenzofurazan (NBD‐F). Chromatographic separation was performed on a Inertsil C₁₈ column (150 mm × 4.6 mm, 5 µm) using isocratic elution by a mobile phase consisting of methanol/water (55:45) at a flow rate of 1.2 mL/min. Amlodipine was used as the internal standard (IS). Fluorescence detector was used operated at 461 nm (excitation) and 517 nm (emission), respectively. The calibration curve was linear over the range of 50–2000 ng/mL. This method was successfully applied to a pharmacokinetic study after oral administration of a dose (150 mg) of nizatidine. Copyright © 2013 John Wiley & Sons, Ltd.     

An HPLC procedure for monitoring penicillin G fermentations

Summary An HPLC procedure for the quantification of the precursor, intermediates, product, and degradation product of penicillin G fermentations is described. The method involves the use of an acetonitrile gradient and direct UV detection with a total run-time of 45 minutes including column regeneration. The technique can be exploited for on-line monitoring of penicillin G fermentations using defined or complex media.