Determination of the enantiomers of 3-tert.-butylamino-1,2-propanediol by high-performance liquid chromatography coupled to evaporative light scattering detection

A method for the separation and quantitation of the enantiomers of 3-tert.-butylamino-1,2-propanediol by high-performance liquid chromatography and evaporative light scattering detection has been developed. Separation of the enantiomers was performed in normal-phase liquid chromatography on a Chiralpak AS chiral stationary phase. The influence of the gas nature, gas pressure and temperature of the drift tube of the evaporative light scattering detector on the detection sensitivity was investigated. The method was validated in terms of linearity, limit of quantitation, accuracy and precision. The enantiomeric excess of (S)-3-tert.-butylamino-1,2-propanediol, used for the industrial synthesis of (S)-timolol, was measured from 0 to 94%.     

Determination of the enantiomers of 3-tert.-butylamino-1,2-propanediol by high-performance liquid chromatography using mass spectrometric detection

The chiral synthesis of beta-blockers such as (S)-timolol requires a sensitive analytical method for the enantioseparation of its intermediate, 3-tert.-butylamino-1,2-propanediol, in the ng/ml range. The method developed is based on on-line normal-phase LC-MS-MS using a chiral stationary phase and an atmospheric pressure chemical ionization (APCI) interface. The MS detection of 3-tert.-butylamino-1,2-propanediol was first optimized with a pneumatically-assisted electrospray interface (ionspray). The APCI interface was then selected for LC-MS-MS because of the incompatibility of electrospray with n-hexane. The method was validated for both enantiomers in the 25-500 ng/ml concentration range.     

甘油催化加氢制备1,2-丙二醇液相产物的气相色谱分析

采用气相色谱-电子轰击离子源质谱(GC-MS)与比较标准品色谱保留值技术,确定了甘油催化加氢制备1,2-丙二醇液相产物的组成。优化了气相色谱-氢火焰离子法(GC-FID)的色谱条件,使主产物与副产物同时达到最佳分离。以正戊醇为内标物,建立了同时检测甘油催化加氢产物中7种醇类化合物的气相色谱分析方法。在分析方法的线性范围内,各化合物加标回收率均在94.4%～112.6%之间,相对标准偏差(RSD)在1.5%～7.2%之间。反应产物中乙醇、异丙醇、丙醇和丙酮醇的方法最小检出限(LOD)小于0.05μg/g,乙二醇、1,2-丙二醇和甘油的LOD分别为0.21μg/g,0.23μg/g和0.25μg/g,线性相关系数均大于等于0.9957。     

Characterisation of iota-carrageenans oligosaccharides with high-performance liquid chromatography coupled with evaporative light scattering detection

Enzymatically digested oligo-iota-carrageenans were separated with liquid chromatography, coupled to evaporative light scattering detection. As expected, compared to oligo-kappa-carrageenans, the additional sulphate group in the neocarrabiose unit of iota-carrageenans significantly modified the separation mechanisms on ion-exchange chromatography, porous graphitic carbon and ion-pair chromatography. The oligomers were then isolated and characterised off-line with electrospray ionisation mass spectrometry in positive-ion mode. The tetrasaccharide, hexasaccharide and octasaccharide that were identified were associated with protonated heptylamine molecules whose number depended on the number of sulphate groups.     

Determination of five major iridoid glucosides in Flos Lonicerae by high-performance liquid chromatography coupled with evaporative light scattering detection

This study presents a new HPLC method using evaporative light scattering detection for the simultaneous determination of live major iridoid glucosides, namely 7-epi-loganin, sweroside, loganin, 7-epi-vogeloside, and secoxyloganin in Flos Lonicerae, an important traditional Chinese medicinal herb. The optimal conditions of separation and detection were achieved on a C18 analytical column with an isocratic mobile phase consisting of methanol-water (30:70, v/v) containing 0.5% acetic acid at the flow-rate of 1.0 ml/min, temperature for the detector drift tube set at 90 degrees C and the nitrogen flow-rate of 2.6 l/min. The limit of detection (S/N = 3) is less than 35.1 microg/ml and the limit of quantification (S/N = 10) is less than 140.1 microg/ml. All calibration curves show good linear regression (r2>0.996) within test ranges. This method provides good reproducibility for the quantification of the major iridoid glucosides in four Lonicera species with overall intra- and inter-day variation of less than 5% and 9%, respectively. The assay was successfully applied to quantify the main iridoid glucosides in the herb and to identify the botanical origin of Flos Lonicerae.     

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.     

Determination of the major isosteroidal alkaloids in bulbs of Fritillaria by high-performance liquid chromatography coupled with evaporative light scattering detection

A new direct HPLC analytical method using evaporative light scattering detection coupled with a low-temperature adapter for the simultaneous determination of the major biologically active isosteroidal alkaloids in Bulbus Fritillariae, a commonly used antitussive traditional Chinese medicinal (TCM) herb, has been developed. The simultaneous separation of eight Fritillaria alkaloids was achieved on a reversed-phase C8 column with an isocratic mobile phase system consisting of acetonitrile-methanol-water (66.5:3.5:30, v/v) containing 0.006% triethylamine. This method provides good reproducibility and sensitivity for the quantification of six major isosteroidal alkaloids, namely peimissine, verticine, verticinone, imperialine, isoverticine and ebeiedine in different Fritillaria species with overall intra- and inter-day precision and accuracy of less than 11% and higher than 90%, respectively. The assay was successfully utilized to quantify the major biologically active alkaloids in five Fritillaria species. The results demonstrate that this method is simple, selective, and suitable for the quality control of this commonly used antitussive TCM herb, Bulbus Fritillariae. reserved.     

Determination of polysorbate 80 in parenteral formulations by high-performance liquid chromatography and evaporative light scattering detection

Drugs that are not very soluble in aqueous formulations are solubilized with surfactants such as polysorbate 80. In order to evaluate the stability of excipient such as polysorbate 80 in drug formulation, a rapid chromatographic methodology is desired; however, polysorbate 80 does not have a strong chromophore for monitoring by absorption spectrometry. A simple and fast method for the analysis of polysorbate 80 in pharmaceutical formulations was developed using high-performance liquid chromatography with evaporative light scattering detection (ELSD). Separation of polysorbate 80 as a single peak was achieved on a C18 column using a methanol/water gradient mobile phase and ELS detection. The method is specific for polysorbate 80 in the formulation as there were no interferences from the drug or other excipients. Precision, recovery, linearity and limit of quantitation/detection experiments gave acceptable results during the evaluation of the method.     

Determination of gentamicin sulfate and related compounds by high-performance liquid chromatography with evaporative light scattering detection

A rapid and simple method for the separation and quantitation of gentamicin sulfate by HPLC coupled with evaporative light scattering detection (ELSD) has been developed. Detection of the different components of gentamicin is problematic because of the lack of UV absorbing chromophore. The use of the universal ELSD avoids the need for sample derivatization or use of specific detector such as pulsed amperometry. Separation was performed on a highpurity C18 125 mm x 4 mm i.d., 3 microm, reversed phase column with 48.5 mM trifluoroacetic acid-methanol (97:3, v/v), as mobile phase at a flow rate of 0.7 ml/min. The influence of the gas nature, gas pressure and temperature of the drift tube of the detector on the detection response was investigated. Optimization was performed with the help of a specific experimental design software. This method allows the determination of the composition in components C1, C1a, C2, C2a and C2b of gentamicin sulfate samples. Mass spectrometry was employed to confirm the ELSD chromatographic profile. The method was validated using methodology described by the International Conference of Harmonization in the field of Medicinal Substances. Commercial samples of different sources were analyzed and results were in good agreement with specifications of both European and United States Pharmacopoeia.     

Determination of rate constants and activation energy of 3-chloro-1,2-propanediol hydrolysis by capillary electrophoresis with electrochemical detection

A method based on capillary electrophoresis with electrochemical detection (CE-ED) to calculate the rate constants and activation energy of 3-chloro-1,2-propanediol (3-MCPD) hydrolysis was described. Effects of several factors, such as the pH value and the concentration of the running buffer, separation voltage, injection time and the potential applied to the working electrode, were investigated to find the optimum conditions. With a 50 cm length of 25 microm diameter fused-silica capillary at a separation of 10 kV, well-defined separation of 3-chloro-1,2-propanediol from glycerol was achieved in 30 mmol/l borax (pH 9.24) within 13 min. Operated in a wall-jet configuration, a 328 microm copper-disk electrode used as the working electrode exhibits good response at 0.65 V (versus SCE) for 3-MCPD and glycerol. The rate constants of 3-MCPD hydrolysis at different temperatures were determined by monitoring the concentration changes of 3-MCPD. At 80, 85 and 90 degrees C, the measured rate constants of 3-MCPD hydrolysis were 3.8 x 10(-3) min(-1), 7.1 x 10(-3) min(-1) and 11.5 x 10(-3) min(-1), respectively. The activation energy for 3-MCPD hydrolysis was calculated to be 118.1 kJ/mol, which is in good agreement with the value in the literature.     

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.     

Molecular sensing of 3-chloro-1,2-propanediol by molecular imprinting

A covalent interaction-based molecularly imprinted polymer (MIP) material for 3-chloro-1,2-propanediol (3-MCPD), a post-testicular anti-fertility agent and possible carcinogen and mutagen in food products containing acid-hydrolyzed vegetable proteins, has been successfully fabricated using 4-vinylphenylboronic acid as the functional monomer. Rebinding assay revealed that the binding constant, K_B, for the receptor sites and non-specific sites are 1.93±0.1×10⁴ and 2.74±0.7×10² M⁻¹, respectively. The estimated number of receptor site, B_max, imprinted is 123.3±3 μmol/g of MIP. The MIP material is able to act as a potentiometric chemosensor for 3-MCPD via increase in Lewis acidity of the receptor sites upon reaction of the arylboronic acid with 3-MCPD to form the more acidic arylboronic acid esters. A simple pH glass electrode is sufficient to monitor the analyte-specific rebinding. In unbuffered aqueous media, linear potentiometric response from 0 to 350 ppm of 3-MCPD can be achieved. The MIP-based chemosensing in a soya sauce matrix has also been attempted. It is found that the dynamic range of the potentiometric chemosensing response of the MIP material is much reduced, probably due to the blocking or deactivation of receptor sites by interferents in soya sauces. Nevertheless, the present work demonstrated the feasibility of using MIP-based chemosensors as semi-quantitative analytical tools for screening purposes in quality control of food products.     

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.     

Separation and quantification of beer carbohydrates by high-performance liquid chromatography with evaporative light scattering detection

An HPLC method with an evaporative light scattering detector was optimized and validated for quantification of carbohydrates in beer. The chromatographic separation was achieved using a Spherisorb NH2, 5 microm chromatographic column and gradient elution with acetonitrile/water. The determinations were performed in the linear range of 0.05-5.0 g/L for fructose, 0.05-5.0 g/L for glucose, 0.05-15.0 g/L for maltose, 0.05-10.0 g/L for maltotriose, and 0.05-5.0 g/L for maltotetraose. The detection limits were 0.005 g/L for fructose, 0.008 g/L for glucose, and 0.01 g/L for maltose, maltotriose, and maltotetraose. The reliability of the method in terms of precision and accuracy was evaluated in three beer matrices, low alcohol beer, 6% alcohol beer, and beer made with part of adjuncts (4.5% alcohol). Relative standard deviations (RSDs) ranged between 1.59 and 5.95% (n = 10), and recoveries ranged between 94 and 98.4%.     

Analysis of neomycin sulfate and framycetin sulfate by high-performance liquid chromatography using evaporative light scattering detection

A rapid and simple method for the determination of main components and related substances of both neomycin sulfate and framycetin sulfate by HPLC and evaporative light scattering detection (ELSD) is described. The method was also used to determine the neomycin B and the sample sulfate content. Detection and quantitation of aminoglycoside antibiotics are problematic because of the lack of UV absorbing chromophore. The use of a universal detector avoids the need for sample derivatization or use of specific detector based on pulsed amperometry described to be difficult in routine assays. Separation was performed with a Polaris C18 150 mm x 4.6 mm i.d., 3 microm reversed-phase column with a solution of 170mM trifluoroacetic acid (TFA) mobile phase at a flow rate of 0.2 mL/min. The chromatographic parameters were optimized with the help of experimental design software. Mass spectrometry (MS) was employed to confirm the ELSD profile. The final method was validated using methodology described by the International Conference of Harmonization in the field of Active Pharmaceutical Ingredients. Commercial samples of different sources were analyzed and results were in good agreement with specifications of the European Pharmacopoeia.     

Determination and quantitation of five cucurbitane triterpenoids in Momordica charantia by reversed-phase high-performance liquid chromatography with evaporative light scattering detection

A simple and specific analytical method for the quantitative determination of five cucurbitane-type triterpenoids isolated from the fruit of Momordica charantia is developed. The triterpenoids present in the fruits of Momordica charantia are separated with an acetonitrile (0.1% acetic acid)-water (0.1% acetic acid)-methanol (0.1% acetic acid) gradient at a flow rate of 0.5 mL/min. The high-performance liquid chromatography separation was performed on a Phenomenex C18 reversed-phase column. By using an evaporative light scattering detector, the main triterpenoids of Momordica charantia could be detected at levels as low as 10 microg/mL. The method was validated for precision, repeatability, and accuracy. The relative standard deviation was between 0.6-4.4%. The method was sensitive, quick, and accurate for the determination of main triterpenes and saponins in Momordica charantia, and can be used for quality control of Momordica charantia and its related dietary supplements.     

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.     

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。     

Determination of phospholipids in milk samples by means of hydrophilic interaction liquid chromatography coupled to evaporative light scattering and mass spectrometry detection

The combined use of the state-of-the-art hybrid mass spectrometers together with high efficient liquid chromatography could surely be a useful tool for such a challenging task, as phospholipids (PLs) analysis. In this research, we used hydrophilic interaction liquid chromatography (150 mm×2.1 mm I.D., 2.7 μm d.p. partially porous column) to achieve the separation of major PLs classes in cow's and donkey's milk samples. Solid-phase extraction (SPE) was performed in order to pre-concentrate minor PLs from non polar lipids (triacylglycerols) and the recovery for the extraction method was assayed on a milk sample, fortified with 5 μg/mL of SM pure standard, and analyzed in triplicate. A value of 89.99% was calculated, with a coefficient of variation (CV%) of 1.93. A 70-min long stepwise gradient of water/acetonitrile afforded baseline separation of PLs classes, at 50 μL/min flow rate. Accurate detection by an ion trap-time of flight (IT-TOF) mass spectrometer (in both positive and negative ionization mode) allowed to fully characterize the distinctive phospholipid profile and fatty acid composition of cow's and donkey's milk, the latter being analyzed for the first time. Evaporative light scattering detection was further employed to attain the quantitative evaluation of major PLs classes identified, by the external calibration method using reference material solutions in the 5-200 μg/mL concentration range. Major difference between the two analyzed samples consisted in the total PLs amount, which in cow's milk was determined as over 20-fold higher than the donkey's.     

Regio-selective synthesis of novel 1-tert-butyl-4-nitro-1H-pyrrole-3-carboxylic acid building block

A convenient, regio-selective synthesis of novel 1-tert-butyl-4-nitro-1H-pyrrole-3-carboxylic acid was developed, utilizing the bulky tert-butyl moiety of 1-tert-butyl-1H-pyrrole to direct selective, un-symmetrical substitutions to the desired 3 and 4 positions.