Post-column addition as a method of controlling triacylglycerol response coefficient of an evaporative light scattering detector in liquid chromatography-evaporative light-scattering detection

The non-linear response is generally the main limitation to the general quantitative use of evaporative light-scattering detection (ELSD). In the particular case of triacylglycerol (TG) analysis, we present a preliminary paper dealing with the use of post-column additives as a means of monitoring the response of such a detector. As TG can form molecular association complexes (ligand-ligate associations) with either cholesterol, urea or silver nitrate, we report the influence of the concentration of each of these chemical compounds in the liquid phase directed towards the ELSD system. The results show that the response coefficient b of the calibration curve either decreases from 1.25-1.30 to 0.51 or increases from 1.25-1.30 to 1.78 according to the nature and concentration of post-column additive. The use of cholesterol as additive, at a discrete concentration, may lead to a linear response curve (b = 1), i.e. to the direct proportionality of ELSD response versus the TG concentration, making quantitative analysis of such solutes easier. On the other hand, to improve sensitivity, the addition of silver nitrate may be chosen for an increase in b value.     

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%.     

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.     

A modification of the internal normalization method with sample splitting

Summary The procedure of sending a part of the sample vapor directly to the detector has been proposed earlier. This permits the quantitative determination of any sample component by relating its peak area to that of the total peak. This method represents a modification of the internal normalization method, with sample splitting. The necessary operating conditions and the applicability range of the method are considered. The dependence of the analytical results on the errors of splitting detector response factors and peak area measurements is discussed. Different types of split systems and flow paths have been tested. The possibilities of this method are demonstrated by determining the total amount of paraffins and cycloparaffins in platforming gasolines enriched in aromatics.     

Determination of Neurotoxin b-ODAP and Non-protein Amino Acids in Lathyrus Sativus by High-Performance Liquid Chromatography with Precolumn Derivatization with 6-Amino quinolyl-N-hydroxysuccinimidyl Carbamate (AQC)

A new method was developed for the quantitative determination of the neurotoxic non-protein amino acid, 3-N-oxalyl-L-2,3-diaminopropionic acid (b -ODAP), its nontoxic a -isomer and other non-protein amino acids in the plant samples of Lathyrus sativus after derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) by reversed-phase high-performance liquid chromatography (HPLC). 2-Amino butyric acid (ABA) was used as an internal standard. The RP HPLC detection limit for both isomers is 1.8 ng with good response linearity. The results are compared with a colorimetric method.     

Calibration of an evaporative light-scattering detector as a mass detector for supercritical fluid chromatography by using uniform Poly(ethylene glycol) oligomers

The quantitativeness of an evaporative light-scattering detector (ELSD) for supercritical fluid chromatography (SFC) was evaluated by using an equimass mixture of uniform poly(ethylene glycol) (PEG) oligomers. Uniform oligomers, in which all molecules have an identical molecular mass, are useful for the accurate calibration of detectors. We calibrated the SFC-ELSD system for various concentrations and molecular masses by using an equimass mixture of PEG oligomers. ELSD not only showed a good linear response to the injected concentration over a wide concentration range, from 10(-4) to 10(-1)g/mL, but also showed a strong dependence on the molecular mass of the solute. By using chromatograms of the equimass mixture of uniform oligomers to calibrate SFC-ELSD, it was possible to determine exact values of not only the average mass but also the molecular-mass distribution for a PEG 1540 sample. The average molecular mass was shifted to a higher value by several percentage points after calibration of the ELSD.     

Fatty acids by high-performance liquid chromatography and evaporative light-scattering detector

A high-performance liquid chromatographic (HPLC) separation method with an evaporative light-scattering detector (ELSD) has been developed for the separation and quantitative analysis of fatty acid methyl esters (FAME) in three different oils. Reverse-phased C18 HPLC separation of 13 FAME is achieved using a methanol/water eluent mixture. The retention times (RT) reflect the elution behavior of these compounds on C18 reversed-phase HPLC. The proposed method is tested on: soybean oil (Glycine max L.) as reference sample, rice bran oil (Oryza sativa L.), pumpkin seed oil (Cucurbita pepo L.) and algal oil (Arthrospira platensis Nordst.).     

A rapid, sensitive high performance liquid chromatographic method for the determination of meropenem in pharmaceutical dosage form, human serum and urine.

A new, simple, precise and rapid high performance liquid chromatographic method was developed for the determination of meropenem in human serum, urine and pharmaceutical dosage forms. Chromatography was carried out on an LC(18) column using a mixture of 15 mM KH(2)PO(4):acetonitrile:methanol (84:12:4; v/v/v), adjusted to pH 2.8 with H(3)PO(4). The proposed method was conducted using a reversed-phase technique, UV monitoring at 307.6 nm and cefepime as an internal standard. The retention times were 5.98 and 7.47 min for cefepime and meropenem, respectively. The detector response was linear over the concentration range of 50-10,000 ng/mL. The detection limit of the procedure was found to be 22 ng/mL. The detection limit for meropenem in human plasma was 108.4 ng/mL and the corresponding value in human urine was 179.3 ng/mL. No interference from endogenous substances in human serum, urine and pharmaceutical preparation was observed. The proposed method is sufficiently sensitive for determination of the concentrations of meropenem and may have clinical application for its monitoring in patients receiving the drug.     

高效液相色谱/挥发激光散射检测器测定奶粉和鸟粪中的甾体化合物

利用反相高效液相色谱／挥发激光散射检测器发展了分析甾体化合物的新方法,采用甲醇和水溶液为冲洗体系,梯度淋洗,12种甾体类化合物在35min内得到很好的分离。方法简单快速,不需衍生,样品经固相萃取预处理,回收率介于84.0％～102.8％之间,响应值和浓度不呈简单的线性关系,在5～50mg／L浓度范围内对峰面积与浓度进行对数线性回归分析,得回归系数大于0.99。     

Determination of aprotinin by titration with bovine trypsin with end-point detection by high-performance liquid chromatography

A method for the determination of aprotinin (bovine pancreatic trypsin inhibitor, BPTI) is described. The procedure involves the formation of the BPTI-trypsin complex in the presence of an excess of BPTI, quantitative separation of the residual BPTI from the mixture by affinity chromatography and identification and evaluation of the residual BPTI by reversed-phase high-performance liquid chromatography. The method is precise with a mean coefficient of variation of 4.0 and 4.3% for intra- and inter-assay runs, respectively, and has a limit of determination of 3.0 micrograms of aprotinin. The proposed method can be applied to commercial samples, even in very dilute solutions, for the standardisation of aprotinin.     

Analysis of Vegetable Oils by High-Performance Liquid Chromatography

A reversed-phase high-performance liquid chromatographic procedure is proposed to monitor the fatty-acid and triglyceride composition of sunflower-seed oil. The procedure uses a column packed with Diasfer-110-C₁₈ (6 m), a 2 : 8 mixture of acetonitrile and acetone as an eluent, and refractometric detection. Analyses that use simple normalization and normalization with correction factors that take into account the difference between calculated refraction coefficients of different triglycerides are compared. It is shown that the proposed procedure can be used for the quantitative determination of total triglycerides in various vegetable oils as well.     

Thin-layer densitometric determination of individual dihydroxyanthraquinone derivatives

Summary A simple thin-layer densitometric method has been developed for the quantitative determination of individual dihydroxyanthraquinone derivatives. Concentration of each compound is expressed in alizarin equivalent which is used as an internal standard. In this method, determinations on standard solutions differed from the known value by ±6%.     

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.     

Analysis of ergot alkaloids by high-performance liquid chromatography : I. Clavinces and simple derivatives of lysergic acid

A method of high-performance liquid chromatography has been developed for the separation and quantitative analysis of a mixture of ergot alkaloids on MicroPak NH₂ columns using isocratic and gradient elution. The mobile phase is diethyl ether-ethanol. Ultraviolet detection is employed at various wavelengths, and the ergot alkaloids are determined using the method of internal normalization.     

基于反相高效液相色谱法构建 QSRR 模型测定萘类及蒽醌类化合物的正辛醇-水分配系数

正辛醇-水分配系数(Kow )是评价药物毒性、活性及跨膜转运等的重要参数,但直接测定法实验过程复杂。本研究采用反相高效液相色谱(RP-HPLC)法,以甲醇-水为流动相,以29种已知 Kow的酸性和中性苯系物及萘类、醌类衍生物为模型化合物,以保留时间两点校正法( DP-RTC)校正保留时间,并由 Snyder-Soczewinski方程得100%水相保留因子(kw ),建立了表观正辛醇-水分配系数 Kow＂与 kw 的定量关系(Quantita-tive structure-retention relationship, QSRR)模型,并对模型进行了内、外部验证。结果表明,不同 pH 下的 QSRR模型线性相关性 R2=0.974~0.976,内部验证( R2cv =0.970~0.973)和外部验证结果(6种验证化合物,1.4%≤相对误差(RE)≤7.9%)令人满意,与考虑了分子结构参数后建立的线性溶剂化能模型(LSER)相比无差异。将建立的 QSRR 模型应用于11种萘类和蒽醌类化合物的 Kow测定,并与软件计算值、摇瓶法实验值比较,结果表明,本方法准确性更高,且简单快捷,可用于快速准确预测复杂混合物体系中组分的 Kow。     

Comparison between charged aerosol detection and light scattering detection for the analysis of Leishmania membrane phospholipids

The performance of charged aerosol detection (CAD) was compared to evaporative light scattering detection (ELSD) for the analysis of Leishmania membrane phospholipid (PL) classes by NP-HPLC. In both methods, a PVA-Sil column was used for the determination of the major Leishmania membrane PLs, phosphatidic acid, phosphatidylglycerol, cardiolipin, phosphatidylinositol, phosphatidylethathanolamine, phosphatidylserine, lysophosphatidylethathanolamine, phosphatidylcholine, sphingomyelin and lysophosphatidylcholine in the same analysis. Although the response of both detection methods can be fitted to a power function, CAD response can also be described by a linear model with determination coefficients (R(2)) ranging from 0.993 to 0.998 for an injected mass of 30 ng to 20.00 microg. CAD appeared to be directly proportional when a restricted range was used and it was found to be more sensitive at lowest mass range than ELSD. With HPLC-ELSD the limits of detection (LODs) were between 71 and 1195 ng and the limits of quantification (LOQs) were between 215 and 3622 ng. With HPLC-CAD, the LODs were between 15 and 249 ng whereas the limits of quantification (LOQs) were between 45 and 707 ng. The accuracy of the methods ranged from 62.8 to 115.8% and from 58.4 to 110.5% for ELSD and CAD, respectively. The HPLC-CAD method is suitable to assess the influence of miltefosine on the composition of Leishmania membrane phospholipids.     

正交投影用于一维重叠色谱峰分辨及背景扣除

对于两组份色谱重叠峰体系,当以小组份色谱峰作为干扰时,本文利用正交投影方法,通过构造正交投影矩阵,利用原始信号及其投影与背景之间的内在关系来确定真实背景,从而将背景干扰予以扣除,使含背景干扰的一维重叠色谱峰得以分辨。模拟研究表明,对于两组份色谱峰重叠体系当以小色谱峰为干扰时本文方法具有很好的分辨效果,对于完全重叠峰,即使其峰分离度为零能得到满意的结果。     

Sucrose as chiral selector for determining enantiomeric composition of phenylalanine by UV-vis spectroscopy and chemometrics

The determination of enantiomeric composition by partial least squares(PLS) modeling of UV-vis spectral data was investigated for samples of phenylalanine(phe) using sucrose as a chiral auxiliary.And a new data preprocess method,reference band normalization,was introduced to eliminate the spectral variations due to the changes of total concentration of phe.The determination coefficient(R~2) and the standard error of calibration set(SEC) of 13 standard samples are 0.9987 and 0.0128 respectively.The standard error of validation set(SECV) of 7 validation samples is 0.0049.The standard error of predict(SEP) of 6 blind samples for evaluating the robustness of the model is 0.0366.The regression model is robust to determine enantiomeric composition when total concentration varied.It is demonstrated that the reference band normalization is a convenient method of compensating for variations in total concentrations without knowing that in advance.     

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.     

A partial least squares based spectrum normalization method for uncertainty reduction for laser-induced breakdown spectroscopy measurements

A bottleneck of the wide commercial application of laser-induced breakdown spectroscopy (LIBS) technology is its relatively high measurement uncertainty. A partial least squares (PLS) based normalization method was proposed to improve pulse-to-pulse measurement precision for LIBS based on our previous spectrum standardization method. The proposed model utilized multi-line spectral information of the measured element and characterized the signal fluctuations due to the variation of plasma characteristic parameters (plasma temperature, electron number density, and total number density) for signal uncertainty reduction. The model was validated by the application of copper concentration prediction in 29 brass alloy samples. The results demonstrated an improvement on both measurement precision and accuracy over the generally applied normalization as well as our previously proposed simplified spectrum standardization method. The average relative standard deviation (RSD), average of the standard error (error bar), the coefficient of determination (R²), the root-mean-square error of prediction (RMSEP), and average value of the maximum relative error (MRE) were 1.80%, 0.23%, 0.992, 1.30%, and 5.23%, respectively, while those for the generally applied spectral area normalization were 3.72%, 0.71%, 0.973, 1.98%, and 14.92%, respectively.