Uncertainty in the determination of glucose in aqueous solutions by high‐performance liquid chromatography with evaporative light scattering detection

The determination of glucose and other carbohydrates is the most widespread chemical analysis that is performed within the industries of food, beverage, forage, biomass, pulp and paper, pharmaceuticals among others. Besides that, sugar refineries need to control their products, by‐products and effluents, and furthermore, glucose in the sucrose refining process, is considered an impurity, which shall be controlled. Being HPLC the most currently instrumental technique used for glucose analysis, the evaporative light scattering detector (ELSD) offers advantages (sensitivity, possibility for operating in gradient mode) over the also used refractive index detector. In this work, an HPLC‐ELSD methodology was optimised and validated, aiming the estimate of the uncertainty associated with the results at low levels of concentration of glucose to be measured. Linearity of the response was obtained in the range of glucose concentrations from 20 to 300 mg/L, with an analysis time of 10 min. The global uncertainty was estimated accordingly to the bottom‐up approach used by Eurachem. It was 13% on average for concentrations from 100 to 300 mg/L. For lower concentrations, uncertainty increased significantly up to 30% in the vicinity of the LOD of the method.     

二醇基柱高效液相色谱-蒸发光散射检测法测定饮料中的糖

采用二醇基柱分离、蒸发光散射检测法测定了饮料中的糖。比较了乙腈-水、二氯甲烷-甲醇两种流动相分析糖的特点。采用二氯甲烷-甲醇为流动相,果糖、葡萄糖、蔗糖、麦芽糖和棉子糖的峰面积与绝对进样量的标准曲线在绝对进样量为1.6μg-40μg时均具有良好的线性关系。该方法对上述5种糖的检测限分别为0.20μg,0.16μg,0.16μg,0.20μg和0.20μg。     

Evaluation of HPSEC‐ELSD method for precise measurement of β‐agarase activity

β‐Agarase activity was monitored by traditional reducing sugar content methods: Somogyi–Nelson's arsenomolybdate, Miller's dinitrosalicylic acid and Kidby and Davidson's ferricyanide methods, as well as by high‐performance size exclusion chromatography coupled with a refractive index detector and an evaporative light scattering detector (ELSD). Calibration curves were established separately for each method to measure the amounts of the neoagaro‐oligosaccharides (NAOS) in the reaction mixtures, which are the products from 1–10 units (U) of β‐agarase cleavage activity on agarose. Product quantities from each monitoring method were compared with the isolated NAOS products. The graphs plotted by agarase activity unit and product concentration clearly displayed that the ELSD method closely followed the results of the isolated products. The percentage deviation of results measured by the five methods away from those of the isolated NAOS product mixture amounted to −13.1–35.1, −21.1–25.5, −27.1–23.81, 6.1–24.3 and 16.2–22.8%, respectively. When the loss during product isolation, about 15–17%, was taken into account, the high precision of the ELSD method was confirmed. HPSEC‐ELSD methods also accurately measured the enzyme kinetics as well as enabling partial identification of oligosaccharides assembled in the NAOS product mixture. This study established the HPSEC‐ELSD system as an alternative method for monitoring agarase activity. Copyright © 2010 John Wiley & Sons, Ltd.     

清开灵注射液HPLC/ELSD指纹图谱建立及质量相关性研究

运用高效液相色谱／蒸发光散射检测器(HPLC／ELSD)建立了复方清开灵注射液的指纹图谱。对其中7种有效成分进行了定量测定,方法学考察结果良好;通过指纹图谱相似度评价软件,对10批清开灵注射液的HPLC／ELSD指纹图谱进行了相似度计算,结果表明：10批样品相似度很好。该方法为中药质量控制提供了一种可以同时实现整体定性、指标成分定量且简便易行的方法模式。     

Comparison of two aerosol-based detectors for the analysis of gabapentin in pharmaceutical formulations by hydrophilic interaction chromatography

Comparison of hydrophilic interaction chromatography (HILIC) columns coupled with an evaporative light scattering detector (ELSD) or charged aerosol detector (CAD) was done for the detection of gabapentin in pharmaceutical formulations. The chromatographic separations were achieved on four HILIC columns: ZIC HILIC, ZIC pHILIC, Luna HILIC, and Atlantis HILIC. Experimental factors such as mobile phase composition, acetonitrile content, and mobile phase pH were evaluated. Validation of method was done in terms of linearity, sensitivity, accuracy, and precision. The performance of ELSD detection method is comparable to that of CAD. The intra-day and inter-day variations were below 1.7% and 3.2% for CAD and 2.8%, and 3.4% for ELSD, respectively. In addition, detection sensitivities of ELSD, CAD, and UV detectors were also compared for HILIC and reversed phase (RP) modes and the highest sensitivities were obtained in the HILIC mode when connected with CAD and ELSD. The developed HILIC aerosol based detection methods were successfully applied to the analysis of gabapentin in commercial tablets and capsules.     

Selective detection of phosphatidylethanol homologues in blood as biomarkers for alcohol consumption by LC‐ESI‐MS/MS

A new validated method for the quantitation of the abnormal phospholipid phosphatidylethanol (PEth)—a biomarker for ethanol uptake—has been developed by LC‐ESI‐MS/MS following miniaturised organic solvent extraction and reversed phase chromatography with phosphatidylbutanol (PBut) as internal standard. PEth homologues with two fatty acid substituents—PEth 18 : 1/18 : 1, PEth 16 : 0/16 : 0—were determined in post‐mortem blood collected from heavy drinkers at autopsy and also in whole blood samples from a volunteer after a single 60 g‐dose of ethanol. Furthermore, PEth 18 : 1/16 : 0 or its isobaric isomer PEth—16 : 0/18 : 1 was detected. In comparison to previous high‐performance liquid chromatography (HPLC) methods with evaporative light scattering detection (ELSD), the LC‐MS/MS‐method is more sensitive—with a limit of detection below 20 ng/ml—and more selective for single PEth homologues, while ELSD has been used for detection of the sum of PEth homologues with approximately 10 times less sensitivity. LC‐MS/MS enables monitoring of PEth homologues as biomarkers for harmful and prolonged alcohol consumption as with HPLC/ELSD earlier, where PEth is measurable in blood only after more than 50 g ethanol daily intake for more than 2 weeks. Because of its higher sensitivity, there is a potential to detect single heavy drinking by LC‐MS/MS, when PEth is formed in very low concentrations. This opens a new field of application of PEth to uncover single or multiple heavy drinking at a lower frequency and with a larger window of detection in blood than before by HPLC/ELSD or by use of other direct markers, e.g. ethyl glucuronide or ethyl sulfate. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaporative light scattering detection: trends in its analytical uses

Evaporative light scattering detection (ELSD) is widely recognized as a universal tool for liquid and supercritical chromatographies. In addition, this detection technique is fully compatible with continuous-flow systems. In fact, the combination of continuous non-chromatographic techniques and ELSD affords the design of simple, reliable systems for extracting qualitative information. This paper reviews instrumental innovations regarding the miniaturization of evaporative light scattering detectors and their uses in micro and capillary liquid chromatography; also, it discusses their increasingly important role in the development of vanguard configurations for sample screening and the determination of total indices without the need for chromatographic separation. Moreover, it compares them with other types of chromatographic detectors in terms of performance. Finally, the potential of ELSD for solving real-life analytical problems arising from the need to meet (bio)chemical information needs is illustrated with various selected applications. Figure New trends in evaporative light scattering detection: recent chromatographic uses and its role in vanguard/rearguard strategies     

正相液相色谱-蒸发光散射法测定食品中的石蜡

采用液相色谱(HPLC)结合蒸发光散射检测器(ELSD)对食品中的石蜡残留物进行了分析和检测.利用正相色谱柱对石蜡和非石蜡组分进行了分离,而无需对石蜡组分进行逐一分离.利用t检验,对构成石蜡的烷烃组分在ELSD检测器上的线性响应进行了显著性误差分析,结果显示,石蜡中烷烃组分具有相似的线性响应.以此为定量依据,实现了食品中石蜡含量的快速定量分析.并对HPLC-ELSD的检测和确证结果与GC-MS法进行了对比.方法的线性范围为10～500 mg/L,相关系数为0.9988;检出限为1 mg/L.以10, 50和100 mg/kg浓度水平添加石蜡时,其回收率在84.6%～105.4%之间,相对标准偏差为5.4%～7.2%.     

Simultaneous determination of five active hydrolysis ingredients from Panax quinquefolium L. by HPLC‐ELSD

An effective method for simultaneous determination of five hydrolysis products of 20 (R)‐dammarane‐3β,6α,12β,20,25‐pentol, 24(R)‐ocotillol, 20(R)‐protopanaxatriol, 20(S)‐panaxatriol and 20(R)‐dammarane‐3β,12β,20,25‐tetrol was developed using high‐performance liquid chromatography with evaporative light scattering detection (HPLC‐ELSD). The hydrolysis products from Panax quinquefolium L. in the stems and leaves, berries, flower buds and roots components were successfully separated on a Kromasil C₁₈ column using methanol and water (83:17, v/v) as mobile phase in 18 min. The parameter for the ELSD was set to a probe temperature of 40°C and the nebulizer for nitrogen gas was adjusted to 3 L/min. All calibration curves showed good linear regression (r > 0.9975) within test ranges. The validation of the method included recovery, linearity, accuracy and precision (intra‐ and inter‐day variation). The accuracy and precision were satisfactory, with the overall intra‐ and inter‐day variation being less than 3.11%, and recoveries of this method were greater than 95.0%. This study developed an effective and rapid method for simultaneous determination of multiple hydrolysis components from Panax quinquefolium L. Copyright © 2010 John Wiley & Sons, Ltd.     

HPLC‐ELSD analysis of spectinomycin dihydrochloride and its impurities

A simple, rapid and reliable reversed‐phase ion‐pair chromatography method by HPLC coupled to an evaporative light scattering detector (ELSD) has been developed to simultaneously determine chloride, spectinomycin and its related substances in a sample. The column was a TSKgel ODS‐100V. The mobile phase was ACN/aqueous solution of 15 mM ammonium acetate adjusted with TFA to pH 3.0 (2:98 v/v), in an isocratic mode. The drift tube temperature was set at 50°C and the nebulizing gas flow rate of air was 3.5 L/min for ELSD detection. Almost all of the reported degradation compounds of spectinomycin such as actinamine, actinospectinoic acid and biosynthesis intermediates such as dihydrospectinomycin diastereoisomers were baseline separated. MS was utilized for the identification of spectinomycin and its seven related substances. The method for the assay of spectinomycin was successfully validated with respect to accuracy, precision (RSD less than 2%), linearity (throughout the linear range 0.025–3 mg/mL, r=0.9993), sensitivity (LOD: 100 ng on column) and robustness. The experimental results demonstrated that the simultaneous determination of chloride, spectinomycin and related substances is feasible in a single run, which suggests applicability in routine assays.     

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.     

Simultaneous determination of eight major steroids from Polyporus umbellatus by high‐performance liquid chromatography coupled with mass spectrometry detections

Polyporus umbellatus is a widely used diuretic herbal medicine. In this study, a high‐performance liquid chromatography coupled with atmospheric pressure chemical ionization–mass spectrometric detection (HPLC‐APCI‐MS) method was developed for qualitative and quantitative analysis of steroids, as well as for the quality control of Polyporus umbellatus. The selectivity, reproducibility and sensitivity were compared with HPLC with photodiode array detection and evaporative light scattering detection (ELSD). Selective ion monitoring in positive mode was used for qualitative and quantitative analysis of eight major components and β‐ecdysterone was used as the internal standard. Limits of detection and quantification fell in the ranges 7–21 and 18–63 ng/mL for the eight analytes with an injection of 10 µL samples, and all calibration curves showed good linear regression (r² > 0.9919) within the test range. The quantitative results demonstrated that samples from different localities showed different qualities. Advantages, in comparison with conventional HPLC–diode array detection and HPLC‐ELSD, are that reliable identification of target compounds could be achieved by accurate mass measurements along with characteristic retention time, and the great enhancement in selectivity and sensitivity allows identification and quantification of low levels of constituents in complex Polyporus umbellatus matrixes. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of carbohydrates in drinks by high-performance liquid chromatography with a dynamically modified amino column and evaporative light scattering detection

A high-performance liquid chromatographic method with a dynamically modified amino column and evaporative light-scattering detector (ELSD) was established for the direct analysis of the carbohydrates in some drinks. A separation column (Zorbax Rx-SIL, 250 mm x 4.6 mm I.D., 5 microm, Hewlett-Packard, USA) which was modified by ethylenediamine and a guard column (Zorbax Rx-SIL, 12.5 mm x 4.6 mm I.D., 5 microm) were used. The mobile phase was a mixture of water-acetonitrile (1:2.6, v/v) containing 0.03% (v/v) ethylenediamine. Regression equations revealed linear relationship (correlation coefficients=0.996-0.999) between the mass of carbohydrates injected and the carbohydrates peak areas detected by ELSD. The detection limits of ELSD (S/N=3) were between 0.2 and 1.2 microg for different carbohydrates. This method is simple and sensitive.     

Simultaneous determination of major bioactive components in Qingkailing injection by high-performance liquid chromatography with evaporative light scattering detection

High-performance liquid chromatography with evaporative light scattering detection (HPLC/ELSD) was established for simultaneous determination of seven major bioactive components of Qingkailing injection including adenosine, geniposide, chlorogenic acid, baicalin, ursodeoxycholic acid, cholic acid, and hyodeoxycholic acid. The proposed method was applied to analyze ten various Qingkailing injections and produced data with acceptable linearity, repeatability, precision and accuracy having a limit of detection (LOD) of 10-50 ng. In comparison with UV detection, HPLC/ELSD permits the determination of non-chromophoric compounds without prior derivatization, and shows good compatibility to the multi-components of complex analytes. The proposed method is a useful alternative for routine analysis in the quality control of traditional Chinese medicine.     

Separation and determination of carbohydrates in drinks by ion chromatography with a self-regenerating suppressor and an evaporative light-scattering detector

Analysis of glucose and other carbohydrates are often performed by use of normal phase HPLC methods with acetonitrile as major eluent coupled with evaporative light-scattering detector (ELSD) or by use of anion-exchange ion chromatography (IC) methods with NaOH as eluent coupled with pulsed amperimetric electrochemical detector. In this work, a novel method for the determination of carbohydrates by IC in conjunction with a self-regenerating suppressor and an ELSD detector was investigated. Three carbohydrates (glucose, fructose, and sucrose) were separated using a KOH eluent generator to avoid the effect of carbon dioxide absorption in the alkaline eluent. Due to the use of the suppressor, non-volatile components were removed and a low salt background (K+ approximately 0.070 microg/mL) can be obtained so the suppressed eluent could directly go into an ELSD detector without obvious interference of inorganic salts. After examining the changes in retention and resolution, an optimized method was established (for IC: using 32 mM KOH as the eluent at a flow rate of 1 mL/min; for ELSD: operated at 95 degrees C, 4.0 bar nitrogen with a gas flow rate of 2.0 L/min) and the linearity, reproducibility, and the limit of detection (LOD) for the three carbohydrates were further evaluated. Regression equations revealed acceptable linearity (correlation coefficients=0.994-0.998) across the working-standard range (100-1000 microg/mL for glucose and sucrose, 150-1000 microg/mL for fructose) and LODs of glucose, fructose, and sucrose were 93, 126, and 90 microg/mL, respectively. This method has successfully been applied to the determination of the three carbohydrates in carbonated cola drinks and fruit juices. The recoveries were between 95 and 113% (n=3) for different carbohydrates.     

Comparative study on major bioactive components in natural, artificial and in-vitro cultured Calculus Bovis

Major bioactive components in various Calculus Bovis, including natural, artificial and in-vitro cultured Calculus Bovis, were comparatively studied. An approach of high-performance liquid chromatography coupled with ultraviolet and evaporative light scattering detections (HPLC/UV/ELSD) was established to simultaneously determinate six bioactive components thereof, including five bile acids (cholic acid, deoxycholic acid, ursodeoxycholic, chenodeoxycholic acid, hyodeoxycholic acid) and bilirubin. ELSD and UV detector were applied to detect bile acids and bilirubin respectively. The assay was performed on a C(18) column with water-acetonitrile gradient elution and the investigated constituents were authenticated by comparing retention times and mass spectra with those of reference compounds. The proposed method was applied to analyze twenty-one Calculus Bovis extraction samples, and produced data with acceptable linearity, precision, repeatability and accuracy. The result indicated the variations among Calculus Bovis samples under different developmental conditions. Artificial and in-vitro cultured Calculus Bovis, especially in-vitro cultured ones, which contain total bioactive constituents no less than natural products and have the best batch-to-batch uniformity, suffice to be used as substitutes of natural Calculus Bovis.     

Effect of the Wavelength of the Laser Beam on the Response of an Evaporative Light Scattering Detector

Abstract

The effects of the wavelength of the laser beam on the response of an evaporative light scattering detector (ELSD) are discussed. Data characterizing the response of the detector and its dependence on the sample size have been collected for six solutes, using a pulsed dye laser as light source. The experimental results suggest that there is little influence of the wavelength on the intensity of the scattered light. On the other hand, the noise decreases in proportion to the wavelength of the incident light beam. Thus, the detection limit (at constant value of the signal to noise ratio) decreases with decreasing wavelengths. The performance of the ELSD improves when a short wavelength is used.     

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.     

Analysis of euphornin in Euphorbia helioscopia L. and its cytotoxicity to mice lung adenocarcinoma cells (LA795)

Euphorbia helioscopia L. has been used as a herbal remedy for cancer in mainland China. Euphornin is one of the main bioactive constituents with the maximal content of Euphorbia helioscopia L. A reversed-phase high-performance liquid chromatography method with evaporative light scattering detection (ELSD) was developed for the analysis of euphornin for better quality control of E. helioscopia L. A good calibration curve in double logarithmic coordinator for euphornin was obtained. The validation study showed high recoveries (>97.0%) and low coefficient of variation (<3.0%). The use of the method on different euphornin extract samples confirmed its effectiveness. It was shown that ELSD was an effective detection method for the analysis of the non-volatile diterpenes from plants used in traditional Chinese medicine. The evaluation of the cytotoxicity of euphornin to mice lung adenocarcinoma cells (LA795) suggested that euphornin was one of the constituents of E. helioscopia L. responsible for the cytotoxicity against carcinoma cells.