In vitro anti-obesity activity by pancreatic lipase inhibition - Simple HPLC approach using EVOO as natural substrate.

BACKGROUND: Pancreatic lipase (PL) is a key lipolytic enzyme in humans for the digestion and absorption of dietary fats. Thereby, PL is a well-recognized target in the management of obesity and its inhibition attracts the interest of researchers globally. The screening of new natural PL inhibitors as alternative strategy to the synthesis of chemical ones represents nowadays a hot topic in research. The main challenge in this matter is the lack of a universal analytical method allowing the monitoring of PL activity and the reliable quantification of lipid digestion products. RESULTS: The (normal phase)-high-performance liquid chromatography-evaporative light scattering detector [(NP)-HPLC-ELSD] method proposed in this work represents a direct and rapid strategy to simultaneously quantify the products obtained from in vitro PL digestion. As one of the main novelties, the triacylglycerol (TAG) fraction from extra-virgin olive oil was selected as natural substrate. The PL activity was measured by monitoring the levels of remaining TAGs and formed free fatty acids (FFAs), using Orlistat as known inhibitor. The method validation confirmed the adequacy of the analytical method for quantitative purposes, showing high recovery percentage values (between 99% and 103%) and low relative standard deviation (RSD%) values (between 2% and 7%) for triolein and oleic acid standard solutions, as well as appreciably low limit of detection (LOD) and limit of quantification (LOQ) values (respectively 58 and 177 ng mL-1 for triolein; 198 and 602 ng mL-1 for oleic acid). Finally, the developed HPLC-ELSD method was successfully applied to evaluate the inhibitory effect of a polyphenolic extract obtained from apple pomace. The results showed a comparable inhibition degree between a 4.0 mg mL-1 apple pomace solution and a 1.0 µg mL-1 Orlistat solution. CONCLUSION: The proposed innovative method reveals highly sensitive and simple to follow the fate of PL digestion, thus opening the way to further investigations in the research of new potentially anti-obesity compounds. © 2022 Society of Chemical Industry.

[Quality evaluation methods of Poria based on yield and content of alkali extracted polysaccharide].

To improve the quality control methods of Poria and develop and utilize its resources fully, alkaline extraction was used in this study to determine the yield and content of alkali-soluble polysaccharides of Poria. The alkali-soluble extracts of Poria were obtained according to the optimum extraction conditions on the basis of single-factor test, and 30 batches of samples were determined. The structure and chemical composition of the alkali-soluble extracts was characterized by high-performance gel permeation chromatography(HPGPC), Fourier transform infrared spectrometry(FT-IR), nuclear magnetic resonance(NMR) spectroscopy and high-performance liquid chromatography(HPLC) with 1-phenyl-3-methyl-5-pyrazolone(PMP-HPLC). The results showed that the content of the alkali-soluble extracts was in the range of 46.98%-73.86%. The main component was ß-(1→3)-glucan, and its molecular mass was about 1.093×10~5. Further, the content of alkali-soluble polysaccharides of Poria was measured by UV-Vis spectrophotometry and HPLC coupled with the evaporative light scattering detector(HPLC-ELSD), and 30 batches of samples were measured. The results indicated that the content of alkali-soluble polysaccharides determined by UV-Vis spectrophotometry was in the range of 73.70%-92.57%, and the content of samples from Hubei province was slightly higher than that from Yunnan province, Anhui province and Hunan province. The content of alkali-soluble polysaccharides determined by HPLC-ELSD was in the range of 51.42%-76.69%, and the samples from Hunan province had slightly higher content than that from the other three provinces. The content determined by UV-Vis spectrophotometry was higher than that by HPLC-ELSD. However, the content determined by HPLC-ELSD was close to that of alkali-soluble extract, which could accurately characterize the content of alkali-soluble polysaccharides in Poria, and the method was simple and repeatable. Therefore, it is recommended that the quantitative analysis method for alkali-soluble extract and alkali-soluble polysaccharides by HPLC-ELSD be used in the quality standards of Poria in Chinese Pharmacopeia.

Carbohydrate analysis of Mortierella alpina by colorimetry and HPLC-ELSD to reveal accumulation differences of sugar and lipid.

OBJECTIVES: To establish reliable methods for the extraction and quantification of the total carbohydrate and intracellular saccharides from Mortierella alpina and study the changes between carbohydrate and lipid in fermentation process. RESULTS: The extraction of mycelia with HCl following a photometric phenol-sulphuric acid reaction was identified as an optimal method for total carbohydrate analysis in Mortierella alpina, which the extraction efficiency performed 1.1-3.6 fold than other five methods. The total carbohydrate content increased from initial 19.26 to 25.86% during early fermentation process and declined gradually thereafter, while the fatty acid was increasing from 8.47 to 31.03%. For separation and qualitative estimation of intracellular saccharides, the acetonitrile/water freeze-thaw method for extraction and Sugar-Pak I column for separation proved to be possible. With the glucose rapidly decreasing at the beginning of growth, the trehalose accumulated rapidly from 1.63 to 5.04% and then decreased slightly but maintain above 4% of dry biomass. CONCLUSIONS: This work established comprehensive carbohydrate extraction and analysis methods of Mortierella alpina and identified the main saccharide in fermentation process which indicated that the accumulation of fatty acids was related to the change of intracellular carbohydrate content.

Determination of Triterpenoids and Phenolic Acids from Sanguisorba officinalis L. by HPLC-ELSD and Its Application.

A novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for simultaneous determination of 11 phenolic acids and 12 triterpenes in Sanguisorba officinalis L. Chromatographic separation was conducted with gradient elution mode by using a DiamonsilTM C18 column (250 mm × 4.6 mm, 5 µm) with the mobile phase of 0.1% acetic acid water (A) and methanol (B). The drift tube temperature of ELSD was set at 70 °C and the nitrogen cumulative flow rate was 1.6 L/min. The method was fully validated to be linear over a wide concentration range (R2 ≥ 0.9991). The precisions (RSD) were less than 3.0% and the recoveries were between 97.7% and 101.4% for all compounds. The results indicated that this method is accurate and effective for the determination of 23 functional components in Sanguisorba officinalis L. and could also be successfully applied to study the influence of processing method on those functional components in Sanguisorba officinalis L.

Chemical Fingerprint Analysis and Quantitative Analysis of Saccharides in Morindae Officinalis Radix by HPLC-ELSD.

A method based on high performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for the quantitative analysis of three active compounds and chemical fingerprint analyses of saccharides in Morindae officinalis radix. Ten batches of Morindae officinalis radix were collected from different plantations in the Guangdong region of China and used to establish the fingerprint. The samples were separated with a COSMOIL Sugar-D column (4.6 mm × 250 mm, 5 µm) by using gradient elution with water (A) and acetonitrile (B). In addition, Trapped-Ion-Mobility (tims) Time-Of-Flight (tims TOF) was used to identify saccharides of Morindae officinalis radix. Fingerprint chromatogram presented 26 common characteristic peaks in the roots of Morinda officinalis How, and the similarities were more than 0.926. In quantitative analysis, the three compounds showed good regression (r = 0.9995-0.9998) within the test ranges, and the recoveries of the method were in the range of 96.7-101.7%. The contents of sucrose, kestose and nystose in all samples were determined as 1.21-7.92%, 1.02-3.37%, and 2.38-6.55%, respectively. The developed HPLC fingerprint method is reliable and was validated for the quality control and identification of Morindae officinalis radix and can be successfully used to assess the quality of Morindae officinalis radix.

Correlations of Fat Content in Human Milk with Fat Droplet Size and Phospholipid Species.

Fat globule size and phospholipid (PL) content in human milk (HM) were investigated. HM was classified into three groups depending on fat content (A < B < C). PL content (mg/100 g HM) was significantly higher in the C group (p < 0.05), indicating its positive relationship with HM fat content. When the PL content was normalized (mg/g fat), that of group A was significantly higher (p < 0.05) and fat droplet size in group C was slightly larger, suggesting that HM fat content is affected by fat droplet numbers to a larger extent than by fat droplet size. A correlation between PC and SM content in HM was observed regardless of fat content, while correlation between PE and either PC or SM increased in the order of C > B > A, hence the composition and content of PL species in HM varied according to its fat content.

Presence of Cholesterol in Non-Animal Organisms: Identification and Quantification of Cholesterol in Crude Seed Oil from Perilla frutescens and Dehydrated Pyropia tenera.

Studies have reported that cholesterol, a molecule found mainly in animals, is also present in some plants and algae. This study aimed to determine whether cholesterol exists in three dehydrated algae species, namely, Pyropia tenera, Saccharina japonica, and Undaria pinnatifida, and in one plant species, namely, Perilla frutescens (four perilla seed oil samples were analyzed). These species were chosen for investigation because they are common ingredients in East Asian cuisine. Gas chromatography-flame ionization detection (GC-FID) analysis found that cholesterol was present in P. tenera (14.6 mg/100 g) and in all four perilla seed oil samples (0.3-0.5 mg/100 g). High-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) also demonstrated that cholesterol was present in P. tenera (14.2 mg/100 g) and allowed the separation of cholesterol from its isomer lathosterol. However, cholesterol could not be detected by HPLC-ELSD in the perilla seed oil samples, most likely because it is only present in trace amounts. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of cholesterol in both P. tenera and perilla seed oil. MRM results further suggested that lathosterol (a precursor of cholesterol) was present in P. tenera.

Detection and quantification of palmolein and palm kernel oil added as adulterant in coconut oil based on triacylglycerol profile.

Economically motivated adulteration of expensive coconut oil with low cost oil, like palm kernel oil and palmolein is difficult to detect and quantify by available methods primarily due to their overlapping physicochemical properties with coconut oil. In the present work, a HPLC method has been developed to detect and quantify the degree of adulteration of coconut oil with palmolein and palm kernel oil based on triglyceride structure. The normalized area percentage of trilaurin (C36) among the three major TAG molecular species dilaurin-monocaprin/myristin-caprylin-laurin (C34), trilaurin (C36) and dilaurin-monomyristin (C38) of coconut oil was chosen as detection index for quantifying degree of adulteration of coconut oil with palm kernel oil, while the area ratio of dipalmitoyl-monoolein: trilaurin was chosen as detection index for quantifying adulteration of coconut oil with palmolein. The RP-HPLC based method developed in the present work is effective with a 2-4% minimum detection limit of adulterant oils and 78-98% detection accuracy depending on the degree of adulteration and types of oil.

[Ingredients changes in Rehmanniae Radix processing based by HPLC-ELSD specific chromatogram].

To establish the HPLC-ELSD specific chromatogram analysis method of Rehmanniae Radix and Rehmanniae Radix Prae-parata, and analyze and compare their chemical compositions, so as to reveal the change regularity of compositions during the proces-sing. By HPLC-ELSD method, the chromatographic column for Prevail Carbohydrate ES(4.6 mm ×250 mm, 5 µm) was adopted, with acetonitrile(A)-water(B) as mobile phase for gradient elution, and the evaporative light-scattering detector was used. A total of 23 batches of Rehmannia Radix samples, and 25 batches of Rehmanniae Radix Praeparata samples and processing dynamic samples were compared. The established method had a great repeatability, precision and stability. Eight common chromatographic peaks were extracted from 23 batches of Rehmanniae Radix samples, 8 common peaks were extracted from 25 Rehmanniae Radix Praeparata, and 7 chromatographic peaks were identified. The composition ratio of Rehmannia Radix was changed greatly during the processing. When the simila-rity≥0.95 and the fructose peak area was more than 2 times of stachyose tetrahydrate or more than 20 times of raffinose, the processing degree conformed to the requirements of empirical identification. The three main oligosaccharides of Rehmanniae Radix were sucrose that was heated to generate fructose and glucose, stachyose tetrahydrate that was heated to generate melibiose, sucrose and fructose, and stachyose tetrahydrate that was heated to generate manninotriose. The change in the index of proportion between monosaccharides and oligosaccharides can be used as the quantitative criterion for the processing quality of Rehmanniae Radix Praeparata.

Development and Validation of an Analytical Method Based on HPLC-ELSD for the Simultaneous Determination of Rosmarinic Acid, Carnosol, Carnosic Acid, Oleanolic Acid and Ursolic Acid in Rosemary.

The safety, efficacy and stability of natural antioxidants have been the focus of research in the food industry, with the aim of rapidly analyzing and controlling the quality of rosemary and its extracts, a novel analytical method involving high-performance liquid chromatography with evaporative light scattering detection (HPLC-ELSD) was developed for the simultaneous determination of rosmarinic acid, carnosol, carnosic acid, oleanolic acid and ursolic acid in rosemary. Chromatographic separation was conducted with gradient elution mode by using a Zorbax SB-C18 column (4.6 mm × 250 mm, 5 µm) with mobile phases of methanol and 0.6% acetic acid. The drift tube temperature of ELSD was 70 °C, and the pressure of nebulizer nitrogen gas was 40 Psi. The method developed has high sensitivity (with limits of detection from 1.3 to 8.6 µg/mL), acceptable linearity over the tested concentrations (with correlation coefficients from 0.991 to 0.999), good repeatability (with intra- and inter-day CV less than 3.1% for all analytes) and satisfactory accuracy (with recovery between 95.5% and 100.8%). The method has been demonstrated as a powerful tool for the functional ingredients analysis and quality control of rosemary and its extracts in a cost- and time-effective manner.

Composition of Sugars in Wild and Cultivated Lingonberries (Vaccinium vitis-idaea L.).

Products of lingonberries are widely used in the human diet; they are also promising beauty and health therapeutic candidates in the cosmetic and pharmaceutical industries. It is important to examine the sugar profile of these berries, due to potential deleterious health effects resulting from high sugar consumption. The aim of this study was to determine the composition of sugars in wild clones and cultivars or lower taxa of lingonberries by HPLC-ELSD method of analysis. Acceptable system suitability, linearity, limits of detection and quantification, precision, and accuracy of this analytical method were achieved. The same sugars with moderate amounts of fructose, glucose, and low amounts of sucrose were found in wild and cultivated lingonberries. Cultivar 'Erntekrone' and wild lingonberries collected from full sun, dry pine tree forests with lower altitude and latitude of the location, distinguished themselves with exclusive high contents of sugars. The changes in the sugar levels during the growing season were apparent in lingonberries and the highest amounts accumulated at the end of the vegetation. According to our findings, lingonberries seem to be an appropriate source of dietary sugars.

[Qualitative and quantitative analysis of major cholic acids in Suis Fellis Pulvis].

This study is to establish a qualitative method for rapid identification of bile acids in Suis Fellis Pulvis based on UHPLC-LTQ-Orbitrap-MS technology,and an HPLC-ELSD internal standard method for the quantitative determination of two glycine-conjugated BAs in Suis Fellis Pulvis.The chromatographic separation of the UHPLC-LTQ-Orbitrap-MS qualitative analysis was achieved on a Waters Acquity UPLC HSS T_3column(2.1 mm×100 mm,1.8µm),with 0.2%formic acid aqueous solution(A)-acetonitrile(B)as mobile phase ingradient elution.Electrospray ionization(ESI)source was applied and operated in negative ion mode.Quantitative analysis was performed at 30℃on a Diamonsil-C_(18)column(4.6 mm×250 mm,5µm).The mobile phase consisted of 0.2%formic acid solution and acetonitrile with gradient elution and the flow rate was 1.0 m L·min~(-1).An ELSD was used with a nitrogen flow-rate of1.4 L·min~(-1)at a drift tube temperature of 60℃and the gain was 1.A total of 14 bile acids in Suis Fellis Pulvis were characterized based on the accurate mass measurements,fragmentation patterns,chromatographic retention times,and reference materials.For the quantitative analysis method,the glycohyodeoxycholic acid and glycochenodeoxycholic acid had good linear relationship in the range of26.52-265.20 mg·L~(-1)(r=0.999 8)and 19.84-198.40 mg·L~(-1)(r=0.999 1),respectively.The average recoveries(n=6)were104.1%and 103.1%,and the RSD were 2.0%and 2.4%.The UHPLC-LTQ-Orbitrap-MS technology provides a fast and efficient qualitative analysis method for identification of bile acids in Suis Fellis Pulvis.The HPLC-ELSD internal standard method is accurate and reliable,which has reference value for the quality control of Suis Fellis Pulvis.

Development and Validation of an HPLC-ELSD Method for the Quantification of 1-Triacontanol in Solid and Liquid Samples.

1-Triacontanol (TRIA) is gaining a lot of interest in agricultural practice due to its use as bio-stimulant and different types of TRIA-containing products have been presented on the market. Up to date, TRIA determination is performed by GC analysis after chemical derivatization, but in aqueous samples containing low amounts of TRIA determination can be problematic and the derivatization step can be troublesome. Hence, there is the need for an analysis method without derivatization. TRIA-based products are in general plant extracts that can be obtained with different extraction procedures. These products can contain different ranges of concentration of TRIA from units to thousands of mg/kg. Thus, there is the need for a method that can be applied to different sample matrices like plant materials and different plant extracts. In this paper we present a HPLC-ELSD method for the analysis of TRIA without derivatization. The method has been fully validated and it has been tested analyzing the content of TRIA in different dried vegetal matrices, plant extracts, and products. The method is characterized by high sensitivity (LOD = 0.2 mg/L, LOQ = 0.6 mg/L) and good precision (intra-day: <11.2%, inter-day: 10.2%) being suitable for routine analysis of this fatty alcohol both for quality control or research purposes.

Determination of Propionylbrassinolide and Its Impurities by High-Performance Liquid Chromatography with Evaporative Light Scattering Detection.

The discovery of brassinolide in 1979, a milestone in brassinosteroids research, has sparked great interest of brassinolide analogs (BLs) in agricultural applications. Among these BLs, propionylbrassinolide has captured considerable attention because it shows plant growth regulating activity with an excellent durability. Two impurities of propionylbrassinolide were isolated and purified by semi-preparative high-performance liquid chromatography (HPLC), and the chemical structures were confirmed. For simultaneous separation and determination of propionylbrassinolide and impurities, an efficient analytical method based on HPLC with evaporative light scattering detector (HPLC-ELSD) was developed. The optimized analysis was performed on a C18 reversed phase column (250 mm × 4.60 mm, 5 µm) with isocratic elution of acetonitrile and water (90:10, v/v) as the mobile phase. The drift tube temperature of the ELSD system was set to 50 °C and the auxiliary gas pressure was 150 kPa. The regression equations demonstrated a good linear relationship (R² = 0.9989-0.9999) within the test ranges. The limits of detection (LODs) and quantification (LOQs) for propionylbrassinolide, impurity 1 and 2 were 1.3, 1.2, 1,3 and 4.3, 4.0, 4.2 mg/L, respectively. The fully validated HPLC-ELSD method was readily applied to quantify the active ingredient and impurities in propionylbrassinolide technical concentrate. Moreover, the optimized separation conditions with ELSD have been successfully transferred to mass spectrometry (MS) detector for LC-MS determination.

[Effect of fermentation on components of bile acids in Arisaema Cum Bile and determination of three kinds of free bile acids in Arisaema Cum Bile].

The aim of this study is to analyze the compositions of main bile acids in fermented and mixed processing products of arisame cum bile from pig bile, and to establish a method for content determination of bile acids in fermented Arisaema Cum Bile. Fermented and mixed processing products were prepared from arisaematis rhizome and arisaematis rhizoma preparatum with pig bile respectively. Then the differences in bile acids compositions between such two kinds of products were compared by high performance liquid chromatography and evaporative light-scattering detector (HPLC-ELSD). With three kinds of free bile acid compositions as the indicators, HPLC-ELSD method was adopted to determine the content of bile acid compositions in fermented product,on Agilent Eclipse XDB C18(4.6 mm×250 mm, 5 µm) chromatographic column, with acetonitrile and 0.1% glacial acetic acid solution (55:45) as mobile phase, at a flow rate of 1 mL·min⁻¹, column temperature of 30 °C, drift tube temperature of 90 °C, and a nitrogen flow rate of 2.2 mL·min⁻¹. The results showed that the bile acids in fermented bile Arisaema were mainly in a free form, while in mixed processing product, the compositions were mainly in a conjugated form. Three kinds of free bile acids, namely porcine cholic acid (HCA), porcine deoxycholic acid (HDCA) and chenodeoxycholic acid (CDCA) in fermented product, showed a good linear relationship in the range of quantification. The average recovery rate was 95.99%-104.3%, complying with the requirements. The results showed that the conjugated bile acids could be transformed into free bile acids during the fermentation of arisaema cum bile. This established method can effectively control the content of bile acids compositions in fermenting arisaema cum bile.

[Determination of three pair of triterpenoid isomers in leaf of Ilex hainanensis by HPLC-ELSD].

The present study is to develop an HPLC-ELSD method for simultaneous determination of three pairs of triterpenoid isomers, Ilexsaponin A1, Ilexhainanoside D, Ilexgenin A, 3ß, 19α-dihydroxyolean-12-ene-24, 28-dioic acid (ilexhainanin E) ursolic acid and oleanic acid in the leaf of Ilex hainanensis, which could provide evidence to the quality control of this herb. The six constituents were measured on a Waters XBridge C18 column (4.6 mm×250 mm, 5 µm), with a mobile phase consisting of methanol (A)- 0.5% formic acid in water (B) at a flow rate of 1.0 mL·min⁻¹ (0-18 min，70%-85% A，18-20 min，85%-95% A；20-35 min，95% A). The carrier gas was N2, and the pressure was 2.8 L·min⁻¹. The drift tube in this experiment were set at 70 °C. The injection volume was 10 µL. The contents of the six triterpenoids in 6 samples were 3.7-8.5, 10.3 -22.1, 2.8-5.9, 7.8-14.1, 2.6-3.8 and 8.8-11.9 mg·g⁻¹, respectively. The established method is proved to be accurate and sensitive for the determination of triterpenoids in Ilicis Hainanensis Folium, and may be used for the quality improvement of this herb.

HPLC-ELSD Quantification and Centrifugal Partition Chromatography Isolation of 8-O-Acetylharpagide from Oxera coronata (Lamiaceae).

INTRODUCTION: Iridoid glycosides possess highly functionalised monoterpenoid aglycon with several contiguous stereocentres. For the most common, they are often present in quantities reaching several percentage of the fresh plant weight, and thus they may be regarded as starting material for the synthesis of a number of new chiral and bioactive molecules. OBJECTIVE: To quantify and to isolate 8-O-acetylharpagide (AH) from several extracts of Oxera coronata R.P.J. de Kok, a Lamiaceae species endemic to New Caledonia, using HPLC-ELSD (evaporative light scattering detector) and centrifugal partition chromatography (CPC). METHODOLOGY: Oxera coronata produces high amounts of AH in leaves, twigs and fruits. Water and methanol extracts of these plant parts were prepared. The content of AH in each extract was quantified by HPLC-ELSD, using acetonitrile-water (+0.1% formic acid) gradient elution. The HPLC method was validated for precision, linearity, limit of detection (LOD), limit of quantification (LOQ) and accuracy. A ternary solvent system ethyl acetate/n-propanol/water (3:2:5, v/v/v) was selected and applied to recover the target compound using Spot CPC from the leaves aqueous extract. RESULTS: HPLC-ELSD analysis followed by CPC purification led to the efficient isolation of AH from O. coronata leaves aqueous extract. CONCLUSION: HPLC-ELSD has proven to be a well-adapted detection and quantification method for iridoid glycosides, while CPC confirmed to be an efficient technique for the isolation of polar compounds. Copyright © 2016 John Wiley & Sons, Ltd.

Simultaneous Determination of Four Triterpenoid Saponins in Aralia elata Leaves by HPLC-ELSD Combined with Hierarchical Clustering Analysis.

INTRODUCTION: Aralia elata leaves are known to have several biological activities, including anti-arrythmia, antitumor, anti-inflammatory, anti-fatigue, antimicrobial and antiviral effects. Our previous study found that triterpenoid saponins from the leaves of A. elata had antitumor effects. Quantification of the triterpenoids is important for the quality control of A. elata leaves. OBJECTIVE: To establish high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD) for the simultaneous determination of four major triterpenoid saponins, including Aralia-saponin IV, Aralia-saponin VI, 3-O-ß-d- glucopyranosyl-(1 â†’ 3)-ß-d-glucopyranosyl-(1 â†’ 3)-ß-d-glucopyranosyl oleanolic acid 28-O-ß-d-glucopyranoside (Aralia-saponin TTP)and Aralia-saponin V. METHODOLOGY: The separation was carried out on a Dikma Diamonsil C18 column (4.6 mm × 250 mm, 5 µm) efficiently with gradient elution consisting of acetonitrile and water. RESULTS: All calibration curves showed good linear regression (R2  > 0.9996) within the ranges of tested concentrations. This validated method was applied to determine the contents of the four major triterpenoid saponins in 53 samples from different regions of northeast China. Hierarchical clustering analysis was first used to classify and differentiate Aralia elata leaves. CONCLUSION: The method developed was successfully applied to analyse four major triterpenoid saponins in Aralia elata leaves which is helpful for quality control of the herb. Copyright © 2017 John Wiley & Sons, Ltd.

Combined Application of UHPLC-QTOF/MS, HPLC-ELSD and 1 H-NMR Spectroscopy for Quality Assessment of DA-9801, A Standardised Dioscorea Extract.

INTRODUCTION: DA-9801, a standardised 50% aqueous ethanolic extract of a mixture of Dioscorea japonica and D. nipponica, is a botanical drug candidate for the treatment of diabetic neuropathy, which finished its US phase II clinical trials recently. An advanced quality control method is needed for further development of DA-9801, considering its high contents of both primary and secondary metabolites. OBJECTIVE: Development of a quality assessment strategy for DA-9801, based on the combination of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H-NMR spectroscopy. METHODS: The method was developed and tested with 15 batch products of DA-9801. The steroidal saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS and were quantified with the validated HPLC-ELSD method. Primary metabolites of DA-9801 were identified and profiled using 1 H-NMR spectrometry. The batch-to-batch equivalence of DA-9801 was tested with the 1 H-NMR spectra using spectral binning, correlation analysis, and principal component analysis. RESULTS: Six major saponins of DA-9801 were tentatively identified by UHPLC-QTOF/MS. Among them, protodioscin and dioscin were quantified by the validated HPLC-ELSD method. Twenty-six metabolites were identified in 1 H-NMR spectra. The similarity between DA-9801 batches could be evaluated with the NMR spectra of DA-9801. The 1 H-NMR method also revealed that two Dioscorea species contributed distinct amino acids to the contents of DA-9801. CONCLUSION: This study validates the effectiveness of UHPLC-QTOF/MS, HPLC-ELSD, and 1 H NMR-combined method for quality control of DA-9801 and its crude materials. Copyright © 2016 John Wiley & Sons, Ltd.

Validation of analytical procedures using HPLC-ELSD to determine six sesquiterpene lactones in Eremanthus species.

Eremanthus species display sesquiterpene lactones with therapeutic potential. We are proposing the development of a new analytical method that has been completely validated to qualify Eremanthus species and its main lactones in raw material using HPLC-ELSD. For the sample preparation, 10.0 mg of powdered Eremanthus leaves was extracted with a 5 mL MeOH/H2 O (9:1 v/v) solution containing scopoletin at 140 µg/mL as the internal standard. For the separation of eight compounds, six of which were lactones, one internal (IS) and one secondary standard were performed utilizing monolithic columns with a nonlinear gradient. The selectivity, stability, precision and matrix effects parameters showed values of RSD of <10%. The six lactones and scopoletin (IS) were recovered with a proportion between 74 and 90% with accuracy represented by error at -25.41%. The linear dynamic range was obtained between 10.0 and 310.0 µg/mL for all compounds with r(2) > 0.9987. The limits of detection and quantitation ranged from 2.00 to 6.79 µg/mL and from 6.00 to 20.40 µg/mL, respectively. Assessing the robustness study, this method can be used in inter-laboratory studies. Using the HPLC-ELSD method, six sesquiterpene lactones including 4ß,5-dihydro-2',3'-dihydroxy-15-deoxy-goyazensolide, goyazensolide, 4ß,5-dihydro-2',3'-epoxy-15-deoxy-goyazensolide, centratherin, 4ß,5-dihydro-15-deoxy-goyazensolide and lychnofolide, were detected and quantified from distinct Eremanthus species, which were collected in different regions.