High-performance Liquid Chromatography-evaporative Light Scattering Detector Research Articles

Determination of the marker compound, chikusetsusaponin IVa in Dolichos lablab L. using HPLC–PDA and HPLC–ELSD

Dolichos lablab L. (Leguminosae) has been used as food or health supplementary food for a long time. The aim of this study is to establish the method of quantitative analysis of chikusetsusaponin IVa for quality assessment of D. lablab using high-performance liquid chromatography (HPLC) with photodiode array (PDA) detector and evaporative light scattering detector (ELSD). Chikusetsusaponin IVa was isolated from water soluble fraction of D. lablab extract. The column for quantitative determination of chikusetsusaponin IVa was SunFire™ C18 column (4.6 × 250 mm, 5 μm) with maintained at 40 °C. Mobile phase was composed 0.1% (v/v) trifluoroacetic acid in water and acetonitrile. The drift tube temperature and nitrogen pressure in ELSD were 60 °C and 360 KPa. The injection volume and flow-rate were 10.0 μL and 1.0 mL/min. The coefficient of determination of calibration curve were 0.9997 (PDA) and 0.9995 (ELSD) and showed good linearity. The recovery of chikusetsusaponin IVa was 100.02–102.02% with relative standard deviation (RSD) value <2.00%. The RSD values of intraday and interday precisions were ≤2.79%. The content of the chikusetsusaponin IVa was detected 0.216 ± 0.004 mg/freeze-dried extract g (PDA) and 0.203 ± 0.004 mg/ freeze-dried extract g (ELSD). The established HPLC assay could be used as a basis for quality assessment of products containing D. lablab and/or D. lablab.

Analysis of astragalosides I, II and IV in some Egyptian Astragalus species and Astragalus dietary supplements using high-performance liquid chromatography/evaporative light scattering detector and non-parametric regression.

GenuTs Astragalus L. is characterised by the presence of cycloartane saponins which have wide biological activities such as antioxidant, immunomodulating' hepatoprotective and anti-inflammatory activities. From these cycloartane saponins are astragalosides I, II and IV which have been regarded as the most important active constituents in Astragalus species. This work describes the quantitative analysis of astragalosides I, II and IV in some Egyptian Astragalus species and Astragalus dietary supplements in a single run by high-performance liquid chromatography/evaporative light scattering detector (HPLC/ELSD) using gradient elution. The method of quantitation adopted in this study is the standard addition method. First and second derivative treatment of the data was performed, and the study presents comparison between two statistical regression methods for handling data; parametric and non-parametric regression methods. Derivative treatment of the chromatographic response data gives improved quantitation of the chromatographic signals. Non-parametric regression of the data using Theil's method is advantageous over the usual least squares method as it assumes that errors could occur in both x- and y-directions and they might not be normally distributed. In addition, it could effectively circumvent any outlier data points. Due to the simplicity and the good accuracy and reproducibility of the suggested methods, they could be used for analysis and quality control of Astragalus species and Astragalus dietary supplements.

Analysis of Free Oligosaccharides (fOS) from Wild-Type Saccharomyces cerevisiae (Baker's Yeast) using Two Different Extraction Methods

The glycomic profiles of free oligosaccharides (fOS) derived from misfolded N- and O-linked glycoproteins and lipid-linked oligosaccharides are important molecular signatures in various biological processes and serve as a readout of functional properties such as glycosidase inhibition. Several glycan extraction methods are available based on different sorbent chemistries that may influence the analytical profiles obtained. However, there is limited availability of studies comparing the effects of sorbent chemistries on glycan profiles. Therefore, in our study, the fOS profiles from wild-type Saccharomyces cerevisiae (Baker’s yeast) extracted using two common methods namely mixed-bed ion-exchange (MBIE) [AG50W-X12 (H+) and AG2-X8 (Cl-)] and reversed-phase (C18) sorbents were compared using total carbohydrate (phenol sulfuric acid) and total protein (bicinchoninic acid, BCA) assays, thin-layer chromatography (TLC) and high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) analyses. MBIE extraction contained higher oligosaccharide and protein (0.26 mg/mL and 1.8 mg/mL) content than C18 extraction (0.11 mg/mL and 0.2 mg/mL). TLC analysis (butanol: ethanol: water = 6:3:1 and 5:4:1) showed the presence of fOS in both the MBIE and C18 extracts based on the detection of orcinol active (UV-inactive) spots. Similar peaks were present in the HPLC-ELSD chromatograms for both extractions methods with MBIE showing higher abundance. Glycan unit (GU) analysis of the dextran standard using HPLC-ELSD showed that the largest possible oligosaccharide structures detected were only di/trisaccharides. Based on all these results, MBIE extraction is a more suitable carbohydrate extraction technique compared to C18 extraction for subsequent profiling and functional studies of fOS.

Evaluation of a sensitive GC–MS method to detect polysorbate 80 in vaccine preparation

Polysorbates are the most versatile and common surfactants used as protein stabilizers. Analysis of residual polysorbate 80 (PS 80) in conjugate vaccine is challenging due to complexity of conjugate matrices and heterogeneity of the structure of the PS 80 analyte. The direct approach using high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) and gas chromatography-mass spectrometry (GC–MS) that is based on oleic acid methyl ester formation followed by transesterification have been evaluated to quantitate residual PS 80 in meningococcal serogroups A, C, W, Y and X bulk conjugates. HPLC-ELSD method was observed to be less sensitive in comparison to the GC-MS method. The GC-MS method showed promise for quantitation of residual polysorbate 80 with advantages of higher sensitivity, simple sample preparation and mass spectral characterization compared to methods reported to literature. The oleic acid methyl ester was solubilized in hexane and injected in GC-MS to separate on highly polar capillary CP-WAX 52 CB Column. The mass spectral analysis showed characteristic ions at m/z 180, 222 and 264. The method was validated with linearity r2 > 0.99 over the concentration range of 0.5 to 100 μg/mL with LOD and LOQ of 0.3 and 0.91 respectively using PS 80 standard. The GC–MS method provides a simple, fast and label free technique for the precise quantitation of residual PS 80 in meningococcal bulk conjugate vaccine sample, achieved with accuracy between 85–105%.

Egg Yolk Phosphatidylethanolamine: Extraction Optimization, Antioxidative Activity, and Molecular Structure Profiling.

Although phosphatidylethanolamine (PE) is an important functional phospholipid, there have been very few reports on its antioxidant activity and corresponding molecular composition. Crude PE was extracted from egg yolk with various solvents, and response surface modeling was carried out to determine the optimum extraction conditions for PE, under which the PE content in extracts reached 58.94 µg/mL. The crude PE was purified using silica gel-based column chromatography. High-purity PE (98%), identified by high-performance liquid chromatography-evaporative light-scattering detector, was obtained using isocratic elution with a mixed solvent (chloroform: methanol: acetic acid=18:5:1) eluent. PE purified from egg yolk exhibited high radical-scavenging activity, determined by electron paramagnetic resonance (EPR). The result was attributed to the high unsaturated fatty acids (83.10%) content in egg yolk PE, and the unsaturated fatty acids were identified as PE-16:0/18:1Δ 9 , PE-16:0/18:2Δ 9,12 , PE-16:0/20:4Δ 5,8,11,14 , PE-18:0/18:1Δ 9 , PE-18:0/18:2Δ 9,12 , and PE-18:0/20:4Δ 5,8,11,14 by MALDI-TOF MS combined with gas chromatography mass spectrometry. PRACTICAL APPLICATION: In this work, an attempt has been made to explore the antioxidant activity of PE that extracted and purified from egg yolk and its corresponding molecular composition. Owing to its plentiful unsaturated fatty acids (83.10%), purified PE from egg yolk exhibited a high radical-scavenging activity that indicated that egg-yolk PE had a strong antioxidantactivity, and it might exert possible beneficial effects on the human health.

Optimization and comparison of GC-FID and HPLC-ELSD methods for determination of lauric acid, mono-, di-, and trilaurins in modified coconut oil

Modified coconut oil (MCO) obtained from the glycerolysis of virgin coconut oil (VCO) and glycerol under various conditions should have different amounts of bioactive fatty acids (FAs) and acylglycerols (AGs). Methods were developed to analyze lauric acid (LA), monolaurin (ML), dilaurin (DL), and trilaurin (TL) in MCO samples using gas chromatography - flame ionization detector (GC-FID) and high performance liquid chromatography - evaporative light scattering detector (HPLC-ELSD). The purpose of this research is to optimize and compare GC-FID and HPLC-ELSD methods for determination of LA, ML, DL, and TL in MCO samples. All the standard curves exhibited good linearity (R2 ≥ 0.9995), except for that of LA analyzed by HPLC-ELSD (R2 = 0.9971). The limits of detection (LODs) and quantification (LOQs) were found to be in the range of 0.033–0.260 mg/ml and 0.099–0.789 mg/ml for the GC-FID method and 0.040–0.421 mg/ml and 0.122–1.277 mg/ml for the HPLC-ELSD method, respectively. The GC-FID method (LOD ≤ 0.033 mg/ml) was more sensitive than HPLC-ELSD method (LOD ≤ 0.421 mg/ml) and showed satisfactory recoveries for LA analysis while HPLC-ELSD method (LOD ≤ 0.040 mg/ml) was more sensitive than GC-FID method (LOD ≤ 0.260 mg/ml) and exhibited acceptable recoveries for TL analysis. Both methods were applied to determine the MCO samples produced under varied conditions for glycerolysis. The results revealed that the developed GC-FID method is suitable for the quantification of LA, ML, and DL while the developed HPLC-ELSD method is appropriate for the determination of ML, DL, and TL. Both developed GC–FID and HPLC–ELSD methods produced reproducible results for the determination of LA, ML, DL, and TL in MCO samples.

Simultaneous HPLC-ELSD determination of sugars and cyclitols in different parts of Phacelia tanacetifolia Benth.

Three cyclitols (allo-inositol, scyllo-inositol and myo-inositol) and seven sugars (xylose, d-mannose, d-glucose, sucrose, D-(+)-turanose, D-(+)-melezitose monohydrate and D-(+)-raffinose pentahydrate) were determined in different morphological parts of lacy phacelia by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method in 30 min. The applied analytical method is rapid, simple, sensitive, low cost, reliable and suitable for profiling of major sugars and cyclitols in Phacelia tanacetifolia Benth. (lacy phacelia) parts. Pressurized liquid extraction and solid phase extraction were used as extraction and purification methods. The obtained results showed significant differences in the quantities and distribution of cyclitols and sugars found in the investigated morphological parts. The quantities of the identified cyclitols in lacy phacelia ranged between 1.07 and 4.68 mg/g of dried plant, while for sugars in the range of 1.07–25.3 mg/g. However, flowers were richer in cyclitols and sugars compared to the seeds.

Structural Related Effects of Natural Steroid Molecules on Cholesterol Crystallization in Model Bile and Ethanol

AbstractThe purpose of the present experiment is to study the different effects of natural steroid molecules (NSM) on the crystallization of cholesterol in model bile and ethanol. Three natural steroids (stigmasterol, β‐sitosterol, and ergosterol) were selected as model molecules. The supernatant and precipitated crystals collected from 95% ethanol and model bile were quantitatively analyzed by high‐performance liquid chromatography‐evaporative light scattering detection (HPLC‐ELSD), powder X‐ray diffraction, and differential scanning calorimetry, as well as scanning electron microscope, were conducted to understand the crystalline properties of a substance. Results show that Stigmasterol and β‐sitosterol can increase the solubility of cholesterol in specific mass ratios under 95% ethanol system, and lower the solubility of cholesterol in model bile. Ergosterol, on the other hand, has no significant effect on cholesterol crystallization. The reason for this phenomenon is that phytosterols are more similar in molecular structure to cholesterol and act as co‐precipitates with cholesterol in both systems.

Characterizing adsorption of associating surfactants on carbonates surfaces

HypothesisThe adsorption of anionic surfactants onto positively charged carbonate minerals is typically high due to electrostatic interactions. By blending anionic surfactants with cationic or zwitterionic surfactants, which naturally form surfactant complexes, surfactant adsorption is expected to be influenced by a competition between surfactant complexes and surfactant-surface interactions. ExperimentsThe adsorption behavior of surfactant blends known to form complexes was investigated. The surfactants probed include an anionic C15-18 internal olefin sulfonate (IOS), a zwitterionic lauryl betaine (LB), and an anionic C13-alcohol polyethylene glycol ether carboxylic acid (L38). An analytical method based on high-performance liquid chromatography evaporative light scattering detector (HPLC-ELSD) was developed to measure three individual surfactant concentrations from a blended surfactant solution. The adsorption of the individual surfactants and surfactant blends were systematically investigated on different mineral surfaces using varying brine solutions. FindingsLB adsorption on calcite surfaces was found to be significantly increased when blended with IOS or L38 since it forms surfactant complexes that partition to the surface. However, the total adsorption of the LB-IOS-L38 solution on dolomite decreased from 3.09 mg/m2 to 1.97 mg/m2 when blended together compared to summing the adsorption values of individual surfactants, which highlights the importance of mixed surfactant association.

Determination of phytochemical composition and antioxidant capacity of 22 old apple cultivars grown in Poland

The basic chemical composition, polyphenols and antioxidant capacity in 22 old apple cultivars grown in Poland were determined. Fruits were analyzed for contents of individual polyphenolics with the ultra-performance liquid chromatography photodiode detector-quadrupole/time-of-flight mass spectrometry (UPLC-PDA-Q/TOF–MS) method, sugar with the high-performance liquid chromatography–evaporative light scattering detector (HPLC-ELSD) method, and antioxidant capacity with the ABTS and FRAP radical method. A total of 29 bioactive compounds, including 26 polyphenolic compounds (7 flavan-3-ols, 2 dihydrochalcones, 4 anthocyanins, 5 phenolic acids, 8 flavonols) and 3 triterpenoids (ursolic, betulinic and oleanolic acids), were identified in fruits. All the apple cultivars were found to be rich in polyphenols [average 2139.21 mg/100 g dry matter (dm)], especially phenolic acid (average 694.12 mg/kg dm), flavan-3-ols (average 1259.80 mg/kg dm), flavonols (average 106.78 mg/kg dm) and triterpenoids (average 2552.20 µg/g dm), particularly ursolic acid (average 2234.50 µg/g dm), with high ABTS and FRAP capacity (average 72.14 and 46.77 µmol Trolox/100 g dm). The apple cultivars were also found to be a good source of pectins (average 1.19%), acids (average 0.67%) and sugars (average 9.11 g/100 g).

Prebiotic potential of xylooligosaccharides derived from finger millet seed coat

ABSTRACTThe present study deals with the enzymatic production of xylooligosaccharides (XOS) from water-soluble xylan (WSX) extracted from finger millet CO9 seed coat and the evaluation of its prebiotic potential. The yield of XOS was 72%, when the WSX of the finger millet seed coat was treated with commercially available xylanase of Thermomyces lanuginosus. The XOS obtained when analyzed using high-performance liquid chromatography–evaporative light scattering detector (HPLC-ELSD) indicated a degree of polymerization (DP) of 2 and Electro Spray Ionization Mass Spectra (ESI-MS) revealed xylobiose as the major sugar. The presence of β-glycosidic linkages of XOS was identified by Fourier Transform Infrared Spectrometer (FTIR) and Nuclear Magnetic Resonance (NMR) studies. The XOS exhibited an antioxidant activity of 75% and prebiotic efficacy. Prebiotic activity in the presence of probiotic strains was strain specific and differences in the prebiotic activity score (PAS) between strains were evident. The cell-free supernatant of Lactobacillus plantarum grown on the XOS from finger millet seed coat showed strong antibacterial activities against four pathogenic microorganisms compared with commercial XOS. Finger millet seed coat represents an abundant and alternative source for the extraction of xylan and its use for prebiotic XOS production.

Phytochemical Composition and Antioxidant Capacity of Seven Saskatoon Berry (Amelanchier alnifolia Nutt.) Genotypes Grown in Poland.

The basic chemical composition, bioactive compounds, and antioxidant capacity of fruits of three new Polish breeding clones (No. 5/6, type S, and type N) and four Canadian cultivars (cvs.) (“Martin”, “Smoky”, “Pembina”, and “Honeywood”) grown in Poland in 2016 were investigated. Fruits were analyzed for their contents of triterpenoids, carotenoids, chlorophylls, and polyphenolics with the ultra-performance liquid chromatography photodiode detector-quadrupole/time-of-flight mass spectrometry (UPLC-PDA-Q/TOF-MS) method, sugar with the high-performance liquid chromatography–evaporative light scattering detector (HPLC-ELSD) method, and antioxidant capacity with the ability to reduce free radical (ABTS) and ferric reducing ability of plasma (FRAP) method. Thirty-eight bioactive compounds, including twenty-eight polyphenolic compounds (four anthocyanins, nine phenolic acids, nine flavonols, and seven flavan-3-ols), four carotenoids, two chlorophylls, and three triterpenoids were identified in the fruits. The fruits of the tested Saskatoon berry genotypes were found to be rich in phenolic compounds (3773.94–6390.36 mg/100 g·dm), triterpenoids (66.55–91.31 mg/kg·dm), and carotenoids (478.62–561.57 mg/kg·dm), with high ABTS and FRAP capacity (10.38–34.49 and 9.66–25.34 mmol·Trolox/100 g·dm, respectively). Additionally, the berries of these genotypes seemed to be a good source of sugar (9.02–19.69 g/100 g), pectins (0.67%–1.33%), and ash (0.59%–0.67%). Some genotypes of Saskatoon berry, especially the clones type S, type N, and cvs. “Honeywood” and “Smoky”, may be selected for their potential applications in commercial cultivation to produce fruits with valuable health-promoting nutritional effects on human health. Additionally, three new genotypes that may offer new functional materials can be recommended for fruit growers.

Quantitative Analysis of Yeast Phospholipids and Sterols by High-Performance Liquid Chromatography-Evaporative Light-Scattering Detection.

Normal-phase high-performance liquid chromatography (HPLC) is a standard method for separating the major lipid classes in an extract. Owing to the absence of a common property like light absorbance in the various lipid classes, evaporative light-scattering detection (ELSD) is the method of choice for qualitative and quantitative lipid detection. In most cases, neutral lipids and polar lipids are separated by different solvent systems, making it necessary to perform multiple analyses. Compared with other techniques like thin-layer chromatography, normal-phase HPLC-ELSD has better reproducibility and allows a higher degree of automation. Here we describe a method for separating and quantifying yeast neutral lipids and glycerophospholipids in one analytical run.

Rapid Quantification and Validation of Lipid Concentrations within Liposomes.

Quantification of the lipid content in liposomal adjuvants for subunit vaccine formulation is of extreme importance, since this concentration impacts both efficacy and stability. In this paper, we outline a high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method that allows for the rapid and simultaneous quantification of lipid concentrations within liposomal systems prepared by three liposomal manufacturing techniques (lipid film hydration, high shear mixing, and microfluidics). The ELSD system was used to quantify four lipids: 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, dimethyldioctadecylammonium (DDA) bromide, and d-(+)-trehalose 6,6′-dibehenate (TDB). The developed method offers rapidity, high sensitivity, direct linearity, and a good consistency on the responses (R2 > 0.993 for the four lipids tested). The corresponding limit of detection (LOD) and limit of quantification (LOQ) were 0.11 and 0.36 mg/mL (DMPC), 0.02 and 0.80 mg/mL (cholesterol), 0.06 and 0.20 mg/mL (DDA), and 0.05 and 0.16 mg/mL (TDB), respectively. HPLC-ELSD was shown to be a rapid and effective method for the quantification of lipids within liposome formulations without the need for lipid extraction processes.

Systematic development of a group quantification method using evaporative light scattering detector for relative quantification of ginsenosides in ginseng products

The determination for the contents of multi-components in ginseng products has come to the fore by demands of in-depth information, but the associated industries confront the high cost of securing pure standards for the continuous quality evaluation of the products. This study aimed to develop a prospective high-performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method for relative quantification of ginsenosides in ginseng products without a considerable change from the conventional gradient analysis. We investigated the effects of mobile phase composition and elution bandwidth, which are potential variables affecting the ELSD response in the gradient analysis. Similar ELSD response curves of nine major ginsenosides were obtained under the identical flow injection conditions, and the response increased as the percentage of organic solvent increased. The nine ginsenosides were divided into three groups to confirm the effect of elution bandwidth. The ELSD response significantly decreased in case of the late eluted ginsenoside in the individual groups under the isocratic conditions. With the consideration of the two important effects, stepwise changes of the gradient condition were carried out to reach a group quantification method. The inconsistent responses of the nine ginsenosides were reconstituted to three normalized responses by the stepwise changes of the gradient condition, and this result actualized relative quantification in the individual groups. The availability was confirmed by comparing the ginsenoside contents in a base material of ginseng products determined by the direct and group quantification method. The largest difference in the determination results from the two methods was 8.26%, and the difference of total contents was only 0.91%.

The inhibition of α-glycosidase and protein tyrosine phosphatase 1B (PTP1B) activities by ginsenosides from Panax ginseng C.A. Meyer and simultaneous determination by HPLC-ELSD

Panax ginseng C.A. Meyer is extensively used as a food additive because of its medicinal and nutritional properties. This study investigated the inhibitory activity of eight ginsenosides against α-glycosidase and protein tyrosine phosphatase 1B (PTP1B). Results showed the anti-hyperglycaemic activities of the eight ginsenosides were in the order of 20(R)-dammarane-3β, 6α, 12β, 20, 25-pentol (25-OH-PPT) > 20(R)-25-methoxydammarane-3β, 12β, 20-tetrol (25-OCH3-PPT) > 20(R)-protopanaxatriol (PPT) > 20(S)-panaxatriol (PT) > 20(R)-dammarane-3β, 12β, 20, 25-tetrol (25-OH-PPD) > 20(R)-25-methoxydammarane-3β, 12β, 20-triol (25-OCH3-PPD) > 20(R)-protopanaxadiol (PPD) > 20(S)-panaxadiol (PD), and 25-OH-PPT exerted stronger inhibitory activity than acarbose and Na3VO4. Meanwhile, simultaneous quantitative estimation results of eight ginsenosides suggested good linear regression within test ranges, precision, accuracy. 25-OH-PPT of the stems (leaves), flowers, and fruits contained 2.69, 3.31 and 7.20%, respectively. High performance liquid chromatography-evaporative light-scattering detector (HPLC-ELSD) method was proven to be simple, fast, and effective.

A Simple HPLC-ELSD Method for Sugar Analysis in Goji Berry

Fructose, glucose, and sucrose were identified and quantified in commercial samples ofLycium barbarumL. fruits (goji berries) by high performance liquid chromatography-evaporative light scattering detector (HPLC-ELSD) method. This study described a rapid, simple, sensitive, selective, and reliable HPLC method suitable for the profiling of major sugars in berries, the evaluation of the nutritional/energetic properties, and assessment of the maturation stage. The proposed analytical method was validated and the results showed good precision, accuracy, and linearity. In all analyzed goji fruits, glucose and fructose were the predominant sugars, while sucrose content was about ten times lower than each monose. It was observed that glucose and fructose were detected in comparable quantities in all considered samples. Quantitative analysis showed that fructose, glucose, and sucrose content ranged from 154.20 to 259.13 mg g−1, from 152.92 to 284.60 mg g−1, and from 13.75 to 36.43 mg g−1, respectively.

Quantitation of the immunological adjuvants, monophosphoryl lipid A and Quil A in poly (lactic-co-glycolic acid) nanoparticles using high performance liquid chromatography with evaporative light scattering detection.

Monophosphoryl lipid A (MPL) and Quil A are two immunological adjuvants commonly used in vaccines. At present no simple, validated methods for the quantification of Quil A and MPL have been previously reported therefore the aim of the current study was to develop a simple, fast and validated method to quantify MPL and Quil A using high performance liquid chromatography evaporative light scattering detection (HPLC-ELSD). The HPLC-ELSD technique was carried out using a ZORBAX Eclipse XDB-C8 column (2.1×50 mm; particle size, 3.5 μm) in an isocratic elution mode at 25 °C. MPL was eluted at a retention time of 1.8 min with methanol-water as the mobile phase and a detector temperature of 75 °C. Quil A was resolved as three peaks with retention times of 4.1, 5.5 and 6.4 min with a detector temperature of 30 °C and with water-acetonitrile and 0.01% formic acid as the mobile phase. The nebulizer pressure and gain were set at 3.5 bar and 10, respectively. Calibration curves plotted for both the adjuvants had an R(2)>0.997. Accuracy, intra- and inter-day precision were within the accepted limits. The limit of detection for MPL and Quil A were calculated as 1.343 and 2.06 μg/mL, respectively. The limit of quantification was 2.445 for MPL and 8.97 μg/mL for Quil A. This analytical method was used to quantify the entrapment and in vitro release of MPL and Quil A in a poly lactic-co-glycolic acid (PLGA) nanoparticle vaccine.

증기화광산란 검출기를 이용한 콩 함유 수용성 탄수화물의 분석

In the present study, a new analytical method was devised for the simultaneous determination of soluble carbohydrates in soybean seeds using high performance liquid chromatography/evaporative light scattering detection (HPLC/ELSD). The limit of quantification (LOQ) for soybean soluble carbohydrates ranged from 5.6～7.6 mg/kg using the HPLC/ELSD method and from 16.2～33.9 mg/kg using the high performance liquid chromatography/refractive index detection (HPLC/RID) method. Therefore, the HPLC/ELSD method was more sensitive than HPLC/RID. The precision values for retention time and peak area of the HPLC/ELSD method were evaluated by inter-day (n=5) and intra-day (n=10) assays using a standard solution. All precision values (CV 2 >0.999. The contents of soluble carbohydrates for the Shingikong (yellow soybean) and Cheongjakong 3 (black soybean) samples were analyzed using the HPLC/RID and HPLC/ELSD methods. The difference in carbohydrate contents between the two detection methods was significant. Carbohydrate contents in the HPLC/ELSD method were higher than those in the HPLC/RID method. Overall, the HPLC/ELSD method showed satisfactory resolution with a favorable LOQ and reproducibility. Therefore, these results indicate that the HPLC/ELSD method may be applied to determine the contents of soluble carbohydrates in soybean seeds and related food stuffs.

Peimisine and peiminine production by endophytic fungus Fusarium sp. isolated from Fritillaria unibracteata var. wabensis

Steroidal alkaloids, as the major biologically active components in Bulbus Fritillariae, possess a variety of toxicological and pharmacological effects on humans. The objective of this work was to determine whether endophytic fungi isolated from fresh bulbs of Fritillaria unibracteata var. wabensis can produce one or more alkaloids like its host plant. Four classical reagents including Wagner's, iodine-potassium iodide, Mayer's and improved Dragendorff's were used for primary screening. Then thin-layer chromatography (TLC) and high performance liquid chromatography-evaporative light scattering detection (HPLC-ELSD) were employed to identify the fermentation products of the selected strains. The results showed that extract from one stain (WBS007) has positive reactions in process of primary screening. A further TLC scan and HPLC-ELSD showed that strain WBS007 had two components with the same TLC relative front (Rf) value and HPLC retention time (RT) as authentic peimisine and peiminine. In addition, strain WBS007 was identified as Fusarium sp. based on phylogenetic analysis of ITS sequences. Thus, strain WBS007 produced the bioactive ingredient peimisine and peiminine, as does its host plant, and could be used for the production of peimisine and peiminine by fermentation.