Simultaneous quantification of terpenelactones and flavonol aglycones in hydrolyzed ginkgo biloba extract by liquid chromatography with inline ultraviolet and evaporative light scattering detection

We report here a liquid chromatography (LC) method with inline ultraviolet/evaporative light scattering (UV/ELS) detection for the simultaneous quantification of the terpenelactones and flavonol aglycones in a single sample of hydrolyzed Ginkgo biloba extract (GBE). The sample is hydrolyzed by a rapid and convenient oven heating method for 1 h at 90 degrees C with 10% hydrochloric acid. The 1 h hydrolysis was found to be equivalent to the 2.25 h reflux treatment for dry powder extract, where total flavonol glycosides were 28.4 and 28.1%, respectively. Acceptable precision was achieved for total terpenelactones [relative standard deviation (RSD) = 4.8%] by ELS detection, and total flavonol aglycones (RSD = 2.3%) by UV detection. The analytical range was 1.5 to 7.3% (w/w) for the individual terpenelactones (ELS) and 2.5 to 15.0% (w/w) for the individual glycosides (UV) calculated from the aglycones quercetin, kaempferol, and isorhamnetin. This improved method allows for the first time high throughput sample preparation coupled with the quantification of the predominant compounds generally used for quality control of GBE in a single assay.     

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.     

微流蒸发光散射检测器与毛细管液相色谱的联用及其在银杏叶提取物分析中的应用

将微流蒸发光散射检测器( μELSD)与毛细管液相色谱(cLC)联用,应用于中药银杏叶提取物及其分散片制剂的分离检测领域。首先对 μELSD仪器参数进行优化。通过调节漂移管温度与载气流量,提高了分析物的响应,并减小了噪声。然后,搭建了cLC-μELSD分离检测平台,其相对常规LC可大大减小实验试剂消耗。流动相A为0.05%(体积分数,下同)三氟乙酸(TFA)水溶液,流动相B为含0.05% TFA的甲醇溶液。最优的洗脱梯度条件为:0~10 min,5%B~25%B;10~25 min,25%B~38%B;25~35 min,38%B;35~40 min,38%B~42%B;40~55 min,42%B~50%B。银杏叶提取物和复杂中药制剂银杏叶提取物分散片都得到了较好的分离,并在其中鉴定到紫外波段几乎无吸收的重要内酯类活性成分白果内酯以及银杏内酯A、B和C。测定了不同厂家银杏叶提取物中萜类内酯洗脱时间的相对标准偏差,结果均不大于2.42%,表明该体系在目标物的分析上具有良好的重现性。实验证明所建立的cLC-ELSD体系在复杂中药体系的分离检测中有良好的应用性。     

Strategies for determination of insulin with tandem electrospray mass spectrometry: implications for other analyte proteins?

Using human insulin (MW 5808 Da) as a model compound, the possible strategies towards optimization of sensitivity and selectivity of measurement by electrospray ionization with a standard triple quadrupole mass spectrometer were investigated. For measurement in selected ion-monitoring (SIM) mode, these strategies involved systematic variation of instrumental parameters and spray pH. In this investigation four different operating modes were used corresponding to positive/negative ionization modes with acidic/basic sprays and pH reversed (hereafter termed 'wrong-way-round' operation); the cone voltage was optimized for each mode of operation. When collision-activated dissociation (CAD) is employed, two additional operation modes are possible: namely, low collision energies (10-35 eV, CAD-l) for the generation of sequence-specific fragments and high collision energies (>80 eV, CAD-h) for the generation of nonspecific fragments. Overall, this results in twelve different modes of operation. Loop-injection of aqueous insulin standards were run for each of the twelve operating modes and measurements made for five different charge states (n = 2-6) observable with our instrument that has an upper mass limit of m/z 4000. The signal/noise (S/N) ratio was optimized for each charge state, resulting in 60 measurements. The best S/N ratios (20 000) were achieved under positive SIM conditions with charge state 6 (m/z 969) and under 'wrong-way-round' negative SIM conditions with charge state 3 (m/z 1935). Lower S/N ratios were observed under positive CAD-h conditions with charge state 5 (m/z 1163, S/N 15 000) and positive CAD-l conditions with charge state 6 (m/z 969, S/N 10 000). All other operating modes gave maximum S/N ratios of 4000. For measurement of insulin standards, the results obtained show SIM to give the best S/N ratio. However, for samples in complex matrices, our general experience suggests CAD to be the preferable operating mode. Consequently, for the development of a quantitative method for proteins in general, it might be advocated that all of the twelve operating modes and all relevant charge states be investigated to find the optimum S/N ratio.     

Linearization of evaporative light scattering detector signal

Recent advances in nebulizer technology of the evaporative light scattering detector (ELSD) allow the evaporation of trichlorobenzene (TCB) at much lower temperatures than the previous instruments, thus avoiding the sample loss in the lower molecular weight domain. Therefore, the new ELSD opens the possibility to correctly evaluate the molecular weight distribution (MWD) of polymers by gel permeation chromatography, after linearizing the ELSD signal intensity in function of concentration. To find the correct exponent parameter for linearization, it is necessary to take into account not only the peak area, but each point of the chromatogram. The evaluation method for this exponent parameter, found to be 1.61 for the analysis in TCB of polyolefin and polystyrene samples, is presented in this study. This value was verified by the excellent correlation found between the obtained MWD of a high-density polyethylene both with the ELSD and with the traditional differential refractive index detector.     

Improving the universal response of evaporative light scattering detection by mobile phase compensation

Mobile phase compensation, first reported for the charged aerosol detector (CAD), was used as a suitable method to overcome problems related to the mobile phase-dependent response of the evaporative light scattering detector (ELSD). Mobile phase compensation was effectively performed both in the flow injection- and in gradient modes. Without compensation, the response factors of the ELSD for six sulfonamide drugs differed by a factor of two when varying the mobile phase composition between 10 and 90% acetonitrile. This change could be effectively eliminated using the technique of mobile phase compensation, where a secondary pump with a reversed gradient was used to provide the detector with a constant composition of the mobile phase. For identical experimental conditions, the ELSD showed a nearly constant, albeit somewhat reduced, response with compensation. This indicates that under such conditions, the ELSD behaved as a concentration-sensitive detector. The analysis of sulfonamides drugs at 0.05% level using gradient UPLC-ELSD separation with mobile phase compensation is demonstrated.     

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.     

Performance evaluation of evaporative light scattering detection and charged aerosol detection in reversed phase liquid chromatography

In pharmaceutical industry ultraviolet (UV) detection is often used as the preferred detection technique in HPLC analysis since most pharmaceutical compounds possess a UV-absorbing chromophore. However, in case the active pharmaceutical ingredient (API) does not have a UV-absorbing chromophore, or if some of the impurities present lack a chromophore, they will not be detected in routine HPLC analysis employing only a UV detector and alternative detection schemes have to be used. Refractive index detection or mass spectroscopy (MS) can be used but these detectors have their intrinsic weaknesses, such as lack of sensitivity or high cost. With the appearance of semi-universal techniques such as evaporative light scattering detection (ELSD), and more recent, charged aerosol detection (CAD), detection of non-UV-absorbing compounds became feasible without having to resort to such complex or costly detection methods. This paper evaluates the different performance characteristics such as sensitivity, linearity, accuracy and precision of both the ELSD and CAD detector coupled to HPLC. One disadvantage of this type of detector is the non-linear response behaviour which makes direct linear regression for making calibration curves inaccurate.     

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.     

指纹图谱法在参麦注射液质控中的应用

 中医药理论和实践要求综合评价中药的质量 ,指纹图谱法是对中药制剂进行综合宏观分析的可行手段之一 ,因此采用反相高效液相建立了参麦注射液的特征指纹图谱。条件 :Hypersil C18(4 6mmi d × 2 5 0mm ,5 μm)反相柱 ,流动相由水 (A)和乙腈 (B)组成 ,B的体积分数在 5 0min内由 5 %线性增长到 95 % ,流速为1 0mL/min ;紫外检测 ,波长为 2 0 2nm。 2 3个特征指纹峰与内标 (联苯 )的峰面积比作为指标 ,结合主成分分析 投影判别法比较了同一厂家不同批次产品和不同厂家同类产品的化学指纹差异。     

Analysis of terpenelactones in Ginkgo biloba by high performance liquid chromatography and evaporative light scattering detection

A reversed phase HPLC method permitting the determination of 5 terpenelactones in Ginkgo biloba, without the need of any sample preparation is presented in this paper. The compounds were successfully separated within 25 min by using a C-12 column, an evaporative light scattering (ELS) detector and a mobile phase comprising of ammonium acetate buffer, methanol and isobutanol. All terpenelactones were detectable at concentrations as low as 20.3 microg/ml. The analysis of G. biloba market products showed remarkable variations in the lactone content, and more than 2 fold differences in the suggested daily doses of the total lactones, from 8.84 mg to 18.28 mg, respectively.     

Comparison of photodiode array,evaporative light scattering,and single-quadrupole mass spectrometric detection methods for the UPLC analysis of pyrolysis liquids

It is important to analyze pyrolysis liquids to evaluate the yield of valuable products as well as unfavorable by-products. This work focuses on choosing detectors for reversed-phase ultra high-performance liquid chromatography analysis of pyrolysis liquids. The linearity, sensitivity, precision, and recovery of photodiode array (PDA) detector, single-quadrupole mass spectrometer (MS), and evaporative light scattering (ELS) detector were compared for the quantitative determination of several typical compounds found in pyrolysis liquids. PDA and MS detectors were found to be suitable for the quantification of furans and phenol derivatives (furfural, vanillin, syringol), but sugars and their derivatives (glucose, xylose, levoglucosan) can be analyzed with MS or ELS detectors.     

Polyketide analysis using mass spectrometry,evaporative light scattering,and charged aerosol detector systems

In this study, a mass spectrometer (MS), an evaporative light scattering detector (ELSD), and a charged aerosol detector (CAD) were used to analyze an erythromycin precursor (termed 6-deoxyerythronolide B). The work highlights the capabilities of each detector to analyze a representative polyketide compound that does not possess a natural chromophore, and presents the first comparison to include a charged aerosol system. Each detector was evaluated based upon limit of detection (LOD), dynamic range, and precision in the context of polyketide analysis. Due to its low LOD, wide dynamic range, and ability to provide molecular weight information, the MS was deemed the best detection option for the analysis of low-concentration, poorly identified polyketide compounds. Alternatively, both the CAD and ELSD systems studied showed better precision and accuracy. The ELSD demonstrated the best precision at 3%, but its LOD was limited to concentrations primarily greater than or equal to 1 mg/L. The Corona CAD demonstrated a LOD (0.012 mg/L) and dynamic range comparable to mass spectroscopy and therefore serves as a more cost-efficient alternative for polyketide production schemes with low titers.     

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.     

A direct method for the separation and quantification of bile acid acyl glycosides by high-performance liquid chromatography with an evaporative light scattering detector

A direct method for the separation and quantification of a series of bile acid acyl glycosides using high-performance liquid chromatography coupled to an evaporative light scattering detector (HPLC-ELSD) is described. Complete separation of each of 15 bile acid acyl 24-alpha-glucosides and their 24-beta-anomers and 24-beta-galactosides was achieved by the stepwise gradient elution mode on a C18 column using a mixture of acetonitrile-methanol (8:2, v/v) and 1% aqueous acetic acid as the mobile phase. 24-beta-Galactosides were always eluted faster than the corresponding 24-beta-glucosides, which eluted after the corresponding 24-alpha-anomers. Calibration curves of different 24-beta-galactosides were linear over a range of 0.2-40 nmol of injected amount and the detection limits (S/N > 3) were from 0.08 to 0.1 nmol. The present HPLC-ELSD method may provide an insight into the separation and quantification of the biologically interesting neutral bile acids.     

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.     

Use of evaporative light scattering detector in the detection and quantification of enantiomeric mixtures by HPLC

Routinely used in our laboratories at analytical scale, an evaporative light scattering detector (ELSD) has proved to be versatile in the detection of enantiomeric resolution using chiral stationary phases by HPLC. Though this kind of detector has been widely used in various domains, its application in enantiomeric resolution has not been discussed in the literature and is found to have very specific features especially in the quantitative perspective. In contrast with the UV detection, the peak area from ELSD for both enantiomers of a racemic mixture may not be the same. This complicates the assessment of the enantiomeric purity of unknown samples. This current work deals with some practical aspects in the detection of enantiomers and in accurate quantitative determination of enantiomeric purity by ELSD. Effects of analyte nature (more precisely molecular weight and volatility), peak shape and peak shape difference between enantiomers on the quantitative integration by ELSD are discussed in connection with the UV-detection results. The calibration for quantitative enantiomeric analysis and its effectiveness are demonstrated.     

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.