High-performance Liquid Chromatography With Evaporative Light Scattering Detection Research Articles

Quantitative lipidomic analysis of plasma and plasma lipoproteins using MALDI-TOF mass spectrometry

Knowledge of the plasma lipid composition is essential to clarify the specific roles of different lipid species in various pathophysiological processes. In this study, we developed an analytical strategy combining high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) and off-line coupling with matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry (MALDI-TOF/MS) to determine the composition of plasma and major lipoproteins at two levels, lipid classes and lipid species. We confirmed the suitability of MALDI-TOF/MS as a quantitative measurement tool studying the linearity and repeatability for triglycerides (TG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). Moreover, data obtained with this method were correlated with other lipid classes and species measurements using currently available technologies. To establish the potential utility of our approach, human plasma very low density- (VLDL), low density- (LDL) and high density- (HDL) lipoproteins from 10 healthy donors were separated using ultracentrifugation, and compositions of nine lipid classes, cholesteryl esters (CE), TG, free cholesterol (FC), PE, phosphatidylinositol (PI), sulfatides (S), PC, lysophosphatidylcholine (LPC) and sphingomyelin (SM), analyzed. In total, 157 lipid species in plasma, 182 in LDL, 171 in HDL, and 148 in VLDL were quantified. The lipidomic profile was consistent with known differences in lipid classes, but also revealed unexpected differences in lipid species distribution of lipoproteins, particularly for LPC and SM. In summary, the methodology developed in this study constitutes a valid approach to determine the lipidomic composition of plasma and lipoproteins.

Fusarium redolens 6WBY3, an endophytic fungus isolated from Fritillaria unibracteata var. wabuensis, produces peimisine and imperialine-3β-d-glucoside

The major biological active ingredients of Bulbus Fritillariae cirrhosae (BFC) are steroidal alkaloids, such as peimisine, imperialine-3β-d-glucoside, and peimine. The bulbus of Fritillaria unibracteata var. wabuensis (FUW) was officially recorded in the National Pharmacopoeia of China (2010 edition) as one of the sources of BFC because of its positive therapeutic effects and few side effects. The endophytic fungus strain 6WBY3 was isolated from the fresh bulbus of FUW that had been cultivated for six years. Based on morphological methods and the phylogenetic analysis of internal transcribed spacer (ITS) sequences, this strain was identified as Fusarium redolens. Using color reaction analysis, high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD), and mass spectrometry (MS), it was demonstrated that F. redolens 6WBY3 could produce peimisine and imperialine-3β-d-glucoside, similar to its host plant. The yields of peimisine and imperialine-3β-d-glucoside were 16.0μg·l−1 and 18.8μg·l−1, respectively, in one week of culture. These results indicate that F. redolens 6WBY3 is a promising candidate for the large scale production of peimisine and imperialine-3β-d-glucoside. In addition, the results from the strain 6WBY3 lay the foundation for further study into the mechanism of Fritillaria alkaloids biosynthesis in fungi.

Fingerprint of Solanum nigrum L. from different habitats using high performance liquid chromatography with evaporative light-scattering detection

Solanum nigrum L. contains a variety of active ingredients. It has been applied broadly in clinical practice. However, the compositions of Solanum nigrum L. from different cultivated areas are quite different. Therefore, it is necessary to set up a standard fingerprint. In this work, a series of Solanum nigrum L. samples were collected from different habitats. A high performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) method was developed to study the differences in quality of these samples. The method was optimized by comparing different columns, gradient or isocratic elution and temperatures of evaporating tube. A Phenomenex C18 column was finally selected for separation with gradient elution. The mobile phases were acetonitrile-20% (v/v) methanol aqueous solution containing 0. 03% (v/v) triethylamine. The temperature of evaporating tube was set at 40 °C. The validation was performed and the results showed that the method had good precision, repeatability and stability. The validated method was applied to detect Solanum nigrum L. from different habitats. The similarities of the acquired chromatograms were analyzed by using the similarity evaluation system for chromatographic fingerprint of traditional Chinese medicine (TCM). The chromatograms with a similarity between 0. 9 and 1. 0 were normalized and fitted to form a standard fingerprint of Solanum nigrum L. Thirteen common peaks were found and eleven peaks were identified with authentic standards. The results showed that remarkable differences were found among Solanum nigrum L. from different habitats. These herbs could be distinguished based on the compositions detected by HPLC-ELSD. This method provided a technology to evaluate the homogeneity and stability of Solanum nigrum L with wide application prospects and practical value.

HPLC-ELSD determination of kanamycin B in the presence of kanamycin A in fermentation broth.

A novel method for the direct determination of kanamycin B in the presence of kanamycin A in fermentation broth using high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was developed. An Agilent Technologies C18 column was utilized, evaporation temperature of 40°C and nitrogen pressure of 3.5 bar, the optimized mobile phase was water-acetonitrile (65:35, v/v), containing 11.6 mm heptafluorobutyric acid (isocratic elution with flow rate of 0.5 mL/min) with the gain 11. Kanamycin B was eluted at 5.6 min with an asymmetry factor of 1.827. The method showed good linearity over the concentration range of 0.05 to 0.80 mg/mL for the kanamycin B (r(2) = 0.9987). The intra-day and inter-day coefficients of variation obtained from kanamycin B were less than 4.3%. Mean recovery of kanamycin B from spiked fermentation broth was 95%. The developed method was applied to the determination of kanamycin B without any interference from other constituents in the fermentation broth. This method offers simple, rapid and quantitative detection of kanamycin B.

Identification of Unknown Impurity of Azelaic Acid in Liposomal Formulation Assessed by HPLC-ELSD, GC-FID, and GC-MS

The identification of new contaminants is critical in the development of new medicinal products. Many impurities, such as pentanedioic acid, hexanedioic acid, heptanedioic acid, octanedioic acid, decanedioic acid, undecanedioic acid, dodecanedioic acid, tridecanedioic acid, and tetradecanedioic acid, have been identified in samples of azelaic acid. The aim of this study was to identify impurities observed during the stability tests of a new liposomal dosage form of azelaic acid that is composed of phosphatidylcholine and a mixture of ethyl alcohol and water, using high-performance liquid chromatography with evaporative light-scattering detector (HPLC-ELSD), gas chromatography–flame ionisation detection (GC-FID), and gas chromatography–mass spectrometry (GC-MS) methods. During the research and development of a new liposomal formulation of azelaic acid, we developed a method for determining the contamination of azelaic acid using HPLC-ELSD. During our analytical tests, we identified a previously unknown impurity of a liposomal preparation of azelaic acid that appeared in the liposomal formulation of azelaic acid during preliminary stability studies. The procedure led to the conclusion that the impurity was caused by the reaction of azelaic acid with one of the excipients that was applied in the product. The impurity was finally identified as an ethyl monoester of azelaic acid. The identification procedure of this compound was carried out in a series of experiments comparing the chromatograms that were obtained via the following chromatographic methods: HPLC-ELSD, GC-FID, and GC-MS. The final identification of the compound was carried out by GC with MS.

Evaluation of sugar content and composition in commonly consumed Korean vegetables, fruits, cereals, seed plants, and leaves by HPLC-ELSD

In the present investigation, evaluation of sugars viz. fructose, galactose, glucose, sucrose, lactose, maltose, and raffinose in commonly consumed raw Korean vegetables, fruits, cereals, seed plants, and leaves has been analyzed using high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). Of the 58 samples analyzed, Onion showed the highest content of fructose (27.74g/100g) and glucose (31.80g/100g) and Chestnuts showed the highest content of sucrose (21.82g/100g). On the other hand, Glutinous sorghum (polished grain), Green tea leaves, and Paddy rice (well-polished rice) showed the lowest content of fructose (0.20g/100g), glucose (0.68g/100g) and sucrose (0.23g/100g), respectively. Glutinous barley (Hopimbori-whole grain) and Green tea leaves showed 0.17g/100g and 0.57g/100g of galactose and lactose respectively. Glutinous barley (Seodunchalbori-polished grain) and Black soybeans (Cheongjaho, dried) showed the highest content of maltose (0.51g/100g) and raffinose (1.82g/100g), respectively. In few samples, galactose, maltose, lactose, and raffinose were detected in trace quantities. A partial least squares discriminant analysis (PLS-DA) was also performed to discriminate the analyzed samples.

Ultrasonic-assisted Micellar Extraction and Cloud-point Pre-concentration of Major Saikosaponins in Radix Bupleuri using High Performance Liquid Chromatography with Evaporative Light Scattering Detection

A new ultrasonic-assisted micellar extraction and cloud-point pre-concentration method was developed for the determination of major saikosaponins, namely saikosaponins -A, -C and -D, in Radix Bupleuri by high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD). The non-ionic surfactant Genapol X-080 (oligoethylene glycol monoalkyl ether) was chosen as the extraction additive and parameters affecting the extraction efficiency were optimized. The highest yield was obtained with 10% (w/v) Genapol X-080, a liquid/solid ratio of 200:1 (mL/g) and ultrasonic-assisted extraction for 40 min. In addition, the optimum cloud-point pre-concentration was reached with 10% sodium sulfate and equilibration at for 30 min. Separation was achieved on an Ascentis Express C18 column (100 4.6 mm i.d., 2.7 ) using a binary mobile phase composed of 0.1% acetic acid and acetonitrile. Saikosaponins were detected by ELSD, which was operated at a drift tube temperature and 3.0 bar nebulizer gas () pressure. The water-based solvent modified with Genapol X-080 showed better extraction efficiency compared to that of the conventional solvent methanol. Recovery of saikosaponins ranged from 93.1 to 101.9%. An environmentally-friendly extraction method was successfully applied to extract and enrich major saikosaponins in Radix Bupleuri.

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 C(18) 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.

Simultaneous Determination of Major Bioactive Components in <i>Qingkailing</i> 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.

Assessment of Phospholipid Malabsorption by Quantification of Fecal Phospholipid

ABSTRACTObjectives:The standard methods for quantifying fat absorption involve extraction of fat from fecal samples with heptane, ether and ethanol. These solvents do not quantitatively recover phospholipids. Malabsorption of dietary and biliary phosphatidylcholine could potentially result in choline deficiency. Therefore, the authors developed a method extracting and quantifying fecal phospholipids.Methods:Fecal samples were collected for 72 hours from 18 children with cystic fibrosis and 10 control children. Fat was extracted first with hexane/diethyl ether/ethanol and then with chloroform/methanol. Total fat was quantitated gravimetrically. Phospholipids in extracted fat were separated and quantified using high‐performance liquid chromatography with evaporative light‐scattering detection (HPLC‐ELSD). Phospholipid quantification was validated with a phosphomolybdate colorimetric assay.Results:The combination of solvent systems used in this study significantly improved total fat (p < 0.05) and phospholipid (p < 0.001) extraction compared with either hexane/diethyl ether/ethanol or chloroform/methanol alone. Fecal phospholipid measured by HPLC‐ELSD was significantly correlated with lipid‐soluble phosphorous using the phosphomolybdate assay (r = 0.75, p < 0.001). This method also allows quantification of fecal phosphatidylcholine and lysophosphatidylcholine.Conclusions:Hexane/diethyl ether/ethanol followed by chloroform/methanol extraction of fecal samples and quantification of phospholipids using HPLC‐ELSD is a new method for investigating phospholipid malabsorption.

Assessment of phospholipid malabsorption by quantification of fecal phospholipid.

The standard methods for quantifying fat absorption involve extraction of fat from fecal samples with heptane, ether and ethanol. These solvents do not quantitatively recover phospholipids. Malabsorption of dietary and biliary phosphatidylcholine could potentially result in choline deficiency. Therefore, the authors developed a method extracting and quantifying fecal phospholipids. Fecal samples were collected for 72 hours from 18 children with cystic fibrosis and 10 control children. Fat was extracted first with hexane/diethyl ether/ethanol and then with chloroform/methanol. Total fat was quantitated gravimetrically. Phospholipids in extracted fat were separated and quantified using high-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD). Phospholipid quantification was validated with a phosphomolybdate colorimetric assay. The combination of solvent systems used in this study significantly improved total fat (p < 0.05) and phospholipid (p < 0.001) extraction compared with either hexane/diethyl ether/ethanol or chloroform/methanol alone. Fecal phospholipid measured by HPLC-ELSD was significantly correlated with lipid-soluble phosphorous using the phosphomolybdate assay (r = 0.75, p < 0.001). This method also allows quantification of fecal phosphatidylcholine and lysophosphatidylcholine. Hexane/diethyl ether/ethanol followed by chloroform/methanol extraction of fecal samples and quantification of phospholipids using HPLC-ELSD is a new method for investigating phospholipid malabsorption.