HPLC-ELSD Method Research Articles

Physicochemical Properties, Fatty Acid Profile and Estimation of Phospholipids by HPLC-ELSD Method in Seeds of <i>Abelmoschus esculentus</i> and <i>Benincasa hispida</i>

Background: Abelmoschus esculentus (Ae) and Benincasa hispida (Bh) are herbal plants has embarked its benefits in ayurveda medicine since history. Chemical compounds present in these two plant parts have numerous health benefits. Aim: In the present study seeds of the selected plants Ae and Bh are evaluated for its physicochemical, pharmacognostical, and analyzing fatty acid profile along with estimating important phospholipids such as Phosphatidyl Choline (PC) and Phosphatidyl Ethanolamine (PE) by HPLC-ELSD method. Methods: PC and PE are phospholipids which are the important components of cell membrane. PC and PE are involved in cell signaling, brain development, signal transduction, cell to cell communication and used for the synthesis of acetylcholine, which is a neurotransmitter. Hence, estimating PC and PE in the seeds of Ae and Bh will serve as an important data, and which will act as a source of memory enhancing components. Total lipids were extracted from the seeds using chloroform and methanol. The extracted lipids were subjected to column chromatography to estimate PC and PE. Using seed powder physicochemical and pharmacognostical values were evaluated. Results: Current study results show optimized physicochemical and pharmacognostical values. Fatty acids such as linoleic, palmitic and oleic acids make up the higher percentage. Importantly we could be able to estimate PC and PE by proposed HPLC-ELSD method, in Ae account for 24.6 % (PC) and 75.4% (PE), but unfortunately, we could not be able to estimate phospholipids in Bh by proposed method. Conclusion: The obtained results will serve as a parameter for the standardization of seeds and fixed oil obtained from seeds, with the estimation of phospholipids, which can be utilized as a potential source in the treatment of dementia, Alzheimer’s disease, Parkinson disease etc.

Lipid loss and compositional change during preparation of simple two-component liposomes

Liposomes are used as model membranes in many scientific fields. Various methods exist to prepare liposomes, but common procedures include thin-film hydration followed by extrusion, freeze-thaw, and/or sonication. These procedures can produce liposomes at specific concentrations and lipid compositions, and researchers often assume that the concentration and composition of their liposomes are similar or identical to what would be expected if no lipid loss occurred. However, lipid loss and concomitant biasing of lipid composition can in principle occur at any preparation step due to nonideal mixing, lipid-surface interactions, etc. Here, we report a straightforward HPLC-ELSD method to quantify the lipid concentration and composition of liposomes and apply that method to study the preparation of simple cholesterol/POPC liposomes. We examine common liposome preparation steps, including vortexing during resuspension, lipid film hydration, extrusion, freeze-thaw, and sonication. We found that the resuspension step can play an outsized role in determining the lipid loss (up to ∼50% under seemingly rigorous procedures). The extrusion step yielded smaller lipid losses (∼10–20%). Freeze-thaw and sonication could both be employed to improve lipid yields. Hydration times up to 60 min and increasing cholesterol concentrations up to 50 mol % had little influence on lipid recovery. Fortunately, even conditions with large lipid loss did not substantially influence the target membrane composition, as long as the lipid mixture was below the cholesterol solubility limit. From our results, we identify best practices for producing maximum levels of lipid recovery and minimal changes to lipid composition during liposome preparation for cholesterol/POPC liposomes.

Greenness assessment and comparison of the developed and validated green HPLC-PDA, HPLC-FLD, and HPLC-ELSD methods for the determination of melatonin in various products using analytical eco-scale, NEMI, GAPI, and AGREE greenness metric tools

Melatonin, recognized as the hormone governing sleep, is an endogenous hormone synthesized by the pineal gland situated in the brain. It orchestrates the sleep-wake cycle and fosters optimal circadian rhythm. Owing to its manifold metabolic advantages, particularly in the realm of circadian rhythm regulation, melatonin finds extensive application in various supplementary formulations. The elimination of hazardous chemical waste in common chromatographic separations has become an essential issue. We present a green, eco-friendly, novel, sensitive, rapid, robust, and three simple methods including the HPLC-PDA, HPLC-FLD, and HPLC-ELSD approach for the simultaneous determination of MEL in various products. The mobile phase of all the HPLC methods consists of only water and an ethanol mixture and is free from any toxic solvents. All the separations were made on a C18 column (5 μm particle size, 250 mm x 4.6 mm id.). LODs were found to be as 1.20 ng mL−1, 0.02 ng mL−1, 1.26 µg mL−1, and LOQs 4.00 ng mL−1, 0.07 ng mL−1, 4.21 µg mL−1 for PDA, FLD and ELSD, respectively. The recovery results were found to be within 98.58–102.35 %. Moreover, the greenness assessment of all the HPLC methods was assessed using the Analytical Eco-scale, NEMI, AGREE, and GAPI. The developed methods have been demonstrated to be applicable in routine analyses for the determination of melatonin in various preparations.

Comparison of ELSD and RID combined with HPLC for simultaneous determination of six rare sugars in food components

Evaporative light scattering detection (ELSD) and refractive index detection (RID) are two methods commonly used in high-performance liquid chromatography (HPLC) to quantitatively analyze sugars. The performance of two detectors for the detection and quantification of six rare sugars in food samples is systematically compared in this paper. Erythritol, allulose, tagatose, mannitol, isomaltulose and trehalose were separated by HPLC on an NH2 chromatographic column using a flow gradient or isocratic composition acetonitrile–water (80:20, v/v) as mobile phase. The analytical signals for six rare sugars were obtained using ELSD and RID. The intra-day and inter-day variations were lower 3.9 % and 3.5 % for ELSD and 2.4 % and 3.6 % for RID, respectively. The limits of detection (LODs) were between 0.02 mg·mL−1 and 0.06 mg·mL−1 for ELSD in the range of 0.06–0.6 mg·mL−1. The LODs of RID were between 0.1 mg·mL−1 and 1.0 mg·mL−1 within a wider dynamic range (0.4–80 mg·mL−1). The optimized method was used to analyze seven samples. Although both methods can be used for the detection of six rare sugars, the selectivity of isomaltulose and the detection results of six rare sugars under the HPLC-RID method are not as good as the HPLC-ELSD method. Therefore, the HPLC-ELSD method is more suitable for the detection of six rare sugars in food.

The development and validation of a novel, green, sustainable and eco-friendly HPLC-ELSD method approach for the simultaneous determination of seven artificial sweeteners in various food products: An assessment of the greenness profile of the developed method with an analytical eco-scale, NEMI, GAPI and AGREE

Minimizing the generation of hazardous chemical waste in routine chromatographic separations has become a critical issue. Here, we introduce a totally green, eco-friendly, novel, sensitive, rapid, robust and simple HPLC-ELSD approach for the simultaneous determination of seven artificial sweeteners which are commonly used in various food samples. The mobile phase was totally free from any toxic solvents or additives. Only water and ethanol were used as the best green alternative solvents for each step of the analysis. Separations were carried out on a C18 column (3.5 μm particle size, 150 mm × 4.6 mm id.) by gradient elution using water–ethanol mixture as a mobile phase. The drift tube of ELSD temperature was adjusted to 50 °C and the N2 gas pressure was adjusted to 350 kPa, the Gain value to 7, and the Filter value to 10 on the HPLC-ELSD system. The developed and validated HPLC-ELSD method provided LOD and LOQ in the range of 1.40–2.23 µg mL−1, 4.67–7.43 µg mL−1 respectively, with recoveries between 98.06 and 102.43%. The method precision (RSD) was found to be lower than 1.0%. The applied HPLC-ELSD method permitted the simultaneous determination of seven artificial sweeteners in under fifteen minutes, consuming only 4.3 mL of a green organic solvent (Ethanol). Moreover, the Greenness assessment of the present HPLC-ELSD method was evaluated using NEMI, Analytical Eco-scale, AGREE and GAPI.

Isolation and quantification of a 17-epi-dammarane triterpenoid rhuslactone from roots of Rhus chinensis and its preventive effects on coronary heart disease and thrombosis in zebrafish

Based on mass spectrometry(MS)-guided separation strategy, compound 1 was obtained from the roots of Rhus chinensis. By comprehensive analysis of high resolution-electrospray ionization-mass spectrometry(HR-ESI-MS), nuclear magnetic resonance(NMR) data, and quantum chemical calculation of NMR(qcc-NMR) parameters, compound 1 was elucidated as rhuslactone, a 17-epi-dammarane triterpenoid with a rare 17α-side chain. An HPLC-ELSD method for its quantification in R. chinensis was established and adopted for the quantification of rhuslactone in different batches of R. chinensis. Rhuslactone displayed a good linear relationship within the range of 0.021 3-1.07 μmol·mL~(-1 )(r=0.997 6), and the average recovery was 99.34% [relative standard deviation(RSD) 2.9%). Moreover, the results of the evaluation test of the preventive effects of rhusalctone on coronary heart disease(CHD) and thrombosis showed that rhuslactone(0.11 nmol·mL~(-1)) significantly alleviated heart enlargement and venous congestion and increased cardiac output(CO), blood flow velocity(BFV), and heart rate, thereby reducing thrombus formation in zebrafish with CHD. The effects of rhuslactone on CO and BFV were superior to that of digoxin(1.02 nmol·mL~(-1)), and its effect on improving heart rate was comparable to that of digoxin. This study provides experimental references for the isolation, identification, quality control, and application of rhuslactone from R. chinensis against CHD. It is worth mentioning that this study has discussed some omissions in the determination of the stereochemistry of C-17 in dammarane triterpenoids in the present coursebook Chemistry of Chinese Medicine and some research papers, that is, the compound may be 17-epi-dammarane triterpenoid. This paper has also proposed steps for the establishment of C-17 stereochemistry.

Head-to-Head Comparison of High-Performance Liquid Chromatography versus Nuclear Magnetic Resonance for the Quantitative Analysis of Carbohydrates in Yiqi Fumai Lyophilized Injection

Carbohydrate analysis can be used as a standard analysis for quality control of industries of plants, foods and pharmaceuticals. Quantitative 1H NMR spectroscopy (qNMR) is an excellent alternative to chromatography-based mixture analysis. However, the application of qNMR in sugar analysis has rarely been reported. In this study, the performance of qNMR in sugar analysis was investigated and compared with the results from HPLC analysis. A head-to-head comparison of qNMR (internal and external standard methods) versus HPLC (PMP pre-column derivatization HPLC, HPLC-RID and HPLC-ELSD) based on quantitative analysis of four carbohydrates (fructose, glucose, sucrose and maltose) in Yiqi Fumai lyophilized injection (YQFM) is presented. Both assays showed similar performance characteristics, including linearity range, accuracy, precision and recovery, and analysis times of less than 30 min/sample. After methodological validation, both qNMR and HPLC have good accuracy, precision and stability. Indeed, the qNMR method is simple, sensitive and rapid in quantifying the four sugars. By analysis of variance (ANOVA) for sugar content with HPLC and qNMR methods, we demonstrated that the two analytical methods had no significant difference and could be used interchangeably for the quantitative analysis of carbohydrates.

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

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. 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 4.0mg mL-1 apple pomace solution and a 1.0μg mL-1 Orlistat solution. 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.

Choice of buffer in mobile phase can substantially alter peak areas in quantification of lipids by HPLC-ELSD

Evaporative light scattering detectors (ELSD) are commonly used with high-performance liquid chromatography (HPLC) to separate and quantify lipids, which are typically not easily detectable by more conventional methods such as UV–visible detectors. In many HPLC-ELSD methods to analyze lipids, a volatile buffer is included in the mobile phase to control the pH and facilitate separation between lipid species. Here, we report an unintended effect that buffer choice can have in HPLC-ELSD analysis of lipids – the identity and concentration of the buffer can substantially influence the resulting ELSD peak areas. To isolate this effect, we use a simple isocratic methanol mobile phase supplemented with different concentrations of commonly used buffers for ELSD analysis, and quantify the effect on peak width, peak shape, and peak area for seven different lipids (POPC, DOPE, cholesterol, sphingomyelin, DOTAP, DOPS, and lactose ceramide). We find that the ELSD peak areas for different lipids can change substantially depending on the mobile phase buffer composition, even in cases where the peak width and shape are unchanged. For a subset of analytes which are UV-active, we also demonstrate that the peak area quantified by UV remains unchanged under different buffer conditions, indicating that this effect is particular to ELSD quantification. We speculate that this ELSD-buffer effect may be the result of a variety of physical phenomenon, including: modification of aerosol droplet size, alteration of clustering of analytes during evaporation of the mobile phase, and mass-amplification or ion-pair effects, all of which could lead to differences in observed peak areas. Such effects would be expected to be molecule-specific, consistent with our data. We anticipate that this report will be useful for researchers designing and implementing HPLC-ELSD methods, especially of lipids.

A high-performance liquid chromatography-evaporative light scattering detection-based quantitative analytical method for polyvinyl alcohol in food supplements with pyrolysis-gas chromatography/mass spectrometry-based verification.

Polyvinyl alcohol (PVOH) is extensively used in agricultural, pharmaceutical, textile, and food industries. A new high-performance liquid chromatography (HPLC) method with evaporative light scattering detection (ELSD) was developed for the quantitative analysis of PVOH products. Pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS) was employed for method verification. The HPLC-ELSD method exhibited acceptable linearity (R2 > 0.99), with limits of detection and quantitation of 19.43 and 58.88μg/mL, respectively. Accuracies of 91.16-102.30% were estimated based on recoveries of the intra- and inter-day tests of PVOH. Repeatability and intermediate precision (%RSD) of 1.23-4.45 and 2.18-6.95, respectively, were obtained. The presence of PVOH in the HPLC peaks was further verified using typical indicators of PVOH in Py-GC/MS analysis, such as acetaldehyde, 2,5-dihydrofuran, benzaldehyde, and crotonaldehyde. This novel HPLC method with Py-GC/MS-based verification can be successfully applied for analyzing PVOH in dietary supplement tablets.

Phospholipid composition analysis of krill oil through HPLC with ELSD: Development, validation, and comparison with 31P NMR spectroscopy

A method wherein a high-performance liquid chromatography (HPLC) system is combined with an evaporative light scattering detector (ELSD) was developed and validated as an alternative to the official phosphorous-31 nuclear magnetic resonance (31P NMR) technique to analyze phospholipids in krill oil. A quadratic calibration curve with a good correlation coefficient (>0.999) in the 100–1000 μg mL−1 range was obtained for each phospholipid. The limit of detection (2–7.5 μg mL−1) of the designed assay for the phospholipids in krill oil was good, and the accuracy (99.6–101.0 %) was excellent. The relative standard deviations for repeatability (<1%) and intermediate precision (2.3 %–6.9 %) were acceptable. The results of the developed HPLC-ELSD method applied to krill oil samples were in agreement with those of 31P NMR spectroscopy. Moreover, statistical comparison indicated no significant difference between two methods. Thus, the HPLC-ELSD method can be applied for analyzing the phospholipid composition of krill oil.

Inulin Determination by an Improved HPLC-ELSD Method

Inulin Determination by an Improved HPLC-ELSD Method

Chemical profiling of Fritillariae thunbergii Miq prepared by different processing methods reveals two new quality markers: Zhebeininoside and imperialine-3-β-D-glucoside

Fritillariae thunbergii Miq (FTM)exhibit versatile biological activities including the significant antitussive and expectorant activities. As a herbal medicine, the therapeutic effects of FTM may be expressed by multi-components which have complicated integration effects on multi-targets. With the time going, the different processing methods of FTM has been changed a lot. Thus，the study described the effect of processing methods to FTM and its quality. Studies were undertaken by using UHPLC-LTQ Orbitrap MS and pharmacodynamic models. All reagents were involved of analytical grade. While a HPLC-ELSD's method has been developed and validated, a certified Quality System is conformed to ICH requirements. The experimental animals followed the animal welfare guidelines. We aimed to found the differences after the different processing methods of FTM, and to demonstrate the changes could be selected as quality control indicators, and established a method for simultaneous determination of these for quality control. we have previously found two new steroidal alkaloids: zhebeininoside and imperialine-3-β-D-glucoside from the different processing methods of FTM, which is the difference between the different processing methods of FTM, mainly on the steroidal alkaloids. The activity analysis of zhebeininoside, imperialine-3-β-D-glucoside, verticine and verticinone showed that the mouse model of cough expectorant has antitussive effect. The positive drug selected was dextromethorphan syrup. The positive group showed biological activity, but the blank group showed nothing. The model group showed illness which means that the model was effective. There are two ways of the mechanism of action of the expectorant action which can make sputum thin, reduce its viscosity, and be easy to cough up, or can accelerate the movement of mucous cilia in the respiratory tract and promote the discharge of sputum. In our study, the content of phenol red was significantly reduced in the administration group. To sum up, our results suggest that zhebeininoside and other three components cloud be selected as quality control indicators, and a method for simultaneous determination of zhebeininoside and other three components was established for quality control.

Method Validation and Rapid Determination of Monosodium Glutamate in Various Food Products by HPLC-Fluorescence Detection and Method Optimization of HPLC-Evaporative Light Scattering Detection Approach without Derivatization.

In this study, an effective, simple and rapid high-performance liquid chromatography (HPLC) using fluorescence (FLD) method was developed and validated for the determination of monosodium glutamate (MSG) in 57 various food samples. Besides, HPLC-Evaporate Light Scattering Detection (ELSD) method was carried out for determination of MSG without derivatization. MSG analysis was performed by derivatization with dansyl chloride at excitation 328, emission 530nm with fluorescence detector. HPLC-FLD method was carried out by using C18 (150mm, 4.6mm, 2.7μm) column with the mobile phase consisting of (Water: Methanol:Glacial Acetic Acid)/(54:45:1,v/v/v). The column temperature was set at 25°C and the flow rate was set at 0.5mLmin-1 with an injection volume 20μL. The results were linear (R2 = 0.9999) with very low quantification limits. The applied method was optimized and the validated parameters such as LOD, LOQ, accuracy, precision, linearity and robustness were calculated. The obtained results were statistically compared with each other. The validated HPLC-FLD method was successfully applied for the analysis of MSG in all of the food samples. Moreover, HPLC-ELSD method was optimized and successfully demonstrated for detect the MSG without derivatization.

Research on the effect of spleen-invigorating and anti-swelling active ingredients in crude and processed coix seed based on Spectrum – Effects relationship combined with chemometrics

Coix seed (CS) is the dry mature seed kernel of Coix lacrma-jobi L. var. mayuen (Roman.) Stapf, which has the effect of spleen-invigorating and anti-swelling. However, research reports on the main active ingredients of CS were minimal. The purpose of this study was to find the main active ingredients that affect the efficacy of CS to invigorate the spleen and reduce swelling through the spectrum-effect relationship, combined with chemometrics, grey relational analysis (GRA) and entropy method, and to compare the differences between the effects of crude and processed CS. First of all, the HPLC-ELSD method was used to establish the chromatographic fingerprint of CS, and 12 batches of CS samples were analyzed through chemometrics in this study. Then, we studied the effect of spleen-invigorating and anti-swelling in CS. Finally, through grey relational analysis and entropy method, the spectrum-effect relationship between the chromatographic fingerprint and the seven pharmacodynamic effect indexes was studied. The results showed that the main pharmacologically active ingredients were 1,3-Dioleoyl-2-palmitoylglycerol (peak 8), 1,2-dilinoleoyl-3-oleoyl-rac-glycerol (peak 2), 1,3-Dipalmitoyl-2-Linolein (peak 5), 1,2-Dilinoleoyl-3-palmitoyl-rac-glycerol (peak 3), 1,2-Dioleoyl-3-linoleoyl-rac-glycerol (peak 4), and glycerol trioleate (peak 7), and the comprehensive efficacy of bran-fried CS was better than that of raw CS. In summary, we have identified the main active ingredients related to the efficacy of CS. As far as we know, this is the first time that the crude and processed CS spectrum-effect relationship has been established and compared, which provides a theoretical basis for subsequent studies on the material basis and molecular mechanism of CS pharmacodynamics.

Similar change laws of content of nine components in four processing methods of Polygoni Multiflori Radix Praeparata

To standardize the processing technology of Polygoni Multiflori Radix Praeparata and stabilize its quality, the similar change laws of Polygoni Multiflori Radix Praeparata with different processing methods and time were analyzed. The processing time of Polygoni Multiflori Radix Praeparata was studied at 24, 32, 40, 48 h, and 4 different processing methods were studied, namely stewed with black bean sauce, steamed, steamed with black bean sauce, and steamed with black bean sauce and rice wine. The content of stilbene glycosides and anthraquinones were determined by HPLC-DAD method. UV method was used to determine the content of polysaccharides, and HPLC-ELSD method was used to determine the monosaccharides and oligosaccharides. The comparative chart of content determination, cluster analysis and entropy weight TOPSIS model was used to find the similar change laws and time interval of different processing methods of Polygoni Multiflori Radix Praeparata. The results demonstrated that around 32 h, the content of nine components in Polygoni Multiflori Radix Praeparata with different processing methods had similar change laws, and the decoction pieces had a high quality, indicating that the four processing methods of Polygoni Multiflori Radix Praeparata are likely to be used as one type of decoction piece with the same name.

Quantification of Nutritional Composition and Some Antinutrient Factors of Banana Peels and Pineapple Skins

Agricultural wastes are by-products generated from growing and processing of agricultural commodities such as vegetables, fruits, meats, poultry and crops. The modernisation of agricultural practises creates huge number of wastes namely animals’ carcass, seeds and skins from crop and also trace of pesticide, along the chain. If these wastes are released without proper disposal procedure, it may cause negative effects to environment and jeopardize human health. Banana and pineapple are amongst the most common crops cultivated in tropical countries. With its bright colour, juicy delicious flesh, and well-studied beneficial compounds, these two fruits are being enjoyed as fresh consumption or in the form of food products like chips and jam. Unfortunately, the peel and the skin are currently being dumped to the landfill as waste. The objective of our study was to evaluate the chemical composition of banana peel and pineapple skin, in order to explore the utilisation of these so-called wastes as food ingredients. The samples were analysed for nutritional composition, anti-nutrients level and sugar profile. Proximate analysis according to AOAC 2000 method were conducted to collect the nutritional composition of samples, antinutrients factors were study via spectrophotometery analysis and sugar profile were achieved by using HPLC-ELSD method. The results showed that ash, moisture, fat, protein were in the acceptable level (7.0±0.14, 7.55±1.48, 13.95±1.62, 5.0±2.82, 67.25±3.80 g/100 g respectively for banana peels and 3.49±0.02, 8.65±0.87, 0.38±0.07, 4.84±1.73 and 83.31±3.49 g/100 g for pineapple skins respectively) and acceptable levels of tannin and phytic acid for both samples. Analysis of sugar profile revealed that these high values agricultural waste contain fructose, glucose and sucrose – potentially being utilised as a good source of sweetners. Finally, we recommend that banana peels and pineapple skins should properly be processed and exploited as a high quality and inexpensive source of food ingredients.

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.

Analysis of quality difference based on Astragalus membranaceus var. mongholicus in genuine region

This work is to establish the fingerprint of Astragalus membranaceus var. mongholicus by HPLC-ELSD method, and to analyze the simulated wildness degree of A. membranaceus var. mongholicus in the genuine region of Inner Mongolia, Ningxia and Gansu. Compared with wild A. membranaceus var. mongholicus, the quality differences of A. membranaceus var. mongholicus in the genuine region were analyzed by identification of chromatographic peaks and similarity evaluation, cluster analysis(CA), principal components analysis(PCA) and orthogonal partial least squares discriminant analysis(OPLS-DA). HPLC fingerprints of A. membranaceus var. mongholicus in different genuine regions are established. The qualitative analysis of mass spectrometry identified 18 components. The similarity evaluation shows that the similarity of 32 batches of A. membranaceus var. mongholicus samples was 0.688-0.993. Among them, the similarity of samples in Shanxi, Inner Mongolia, Ningxia is 0.688-0.993, 0.835-0.989, 0.934-0.988, respectively and the similarity of samples in Gansu is 0.729-0.876 except No. 25 sample. The results of CA show that the samples of A. membranaceus var. mongholicus can be grouped into four categories according to the production area except the No. 11 and No. 25 samples. The results of PCA indicate that 32 batches of A. membranaceus var. mongholicus samples can be clustered according to quality and origin, and the quality of A. membranaceus var. mongholicus in Inner Mongolia is the closest to the wild breed. The results of OPLS-DA indicate that there are six components that can distinguish the wild and domestic A. membranaceus var. mongholicus, which are malonylastragaloside Ⅰ, astragaloside Ⅰ, calycosin-7-O-β-D-glycoside-6″-O-malonate, calycosin-7-O-β-D-glycoside, formononetin-7-O-β-D-glycoside-6″-O-malonate, and astrapterocarpan-3-O-β-D-glycoside-6″-O-malonate. The established method can be used to analyze differences between A. membranaceus var. mongholicus origin and planting environment, and can provide references for the protection and replacement of wild A. membranaceus var. mongholicus resources, and the cultivation, processing and production of A. membranaceus var. mongholicus.

A Reliable HPLC-ELSD Method for Determination of Cholesterol, Phosphatidylcholine, Lysophosphatidylcholine Content and the Stability of a Liposomal Formulation

Background:Liposomes continue to play an important role in drug delivery research due to their ability to improve transport and targeting of a wide range of active molecules. Analysis of liposomal components is a key point in the characterization and evaluation of formulation stability. The aim of this work was to develop and validate an HPLC-ELSD method for the characterization and quality control of liposomes.Methods:HPLC-ELSD method was validated by assessing selectivity, linearity, precision, accuracy, limit of detection and quantitation. The mobile phase consisted of a 0.1% (v/v) of trifluoroacetic acid (TFA) and methanol in gradient elution. Initial rate was 20:80 (0.1% TFA: methanol), with a ramp reaching 100% methanol. HPLC-MS/MS was used to confirm the presence of the fatty acid mixture in the analyzed lipids, as well as sub-products generated under pre-determined conditions in the stability study.Results:A HPLC-ELSD method has been developed to detect and measure cholesterol, phosphatidylcholine and lysophosphatidylcholine. High specificity, sensitivity and linearity within the predetermined range for all the compounds analyzed (R2&gt;0.99) were obtained. Accuracy and precision results for all the compounds were within the acceptance limit of ≤5% and 90-110%, respectively. Mass spectrometry results showed complementary information about the phospholipid composition to evaluate the degree of degradation of liposomes over different storage conditions.Conclusion:The method was successfully applied as a quality control tool for the analysis of a wide range of lipids, present in liposomal formulations. HPLC-MS/MS was used to ensure complete elucidation of the lipid components and the detected lyso-forms.