Novel EDTA mediated ethanol protein precipitation method and the application for polysorbate quantification in high protein concentration biopharmaceuticals.

Non-ionic surfactants such as Polysorbate 20/ 80 (PS20/ PS80), are commonly used in protein drug formulations to increase protein stability by protecting against interfacial stress and surface absorption. Polysorbate is susceptible to degradation which can impact product stability, leading to the formation of sub-visible and/or visible particles in the drug product during its shelf-life, affecting patient safety and efficacy. Therefore, it is important to monitor polysorbate concentration in drug product formulations of biotherapeutic drugs. The common method for measuring polysorbate concentration in drug product formulations uses mixed mode ion exchange reversed phase HPLC (MAX) coupled to evaporative light scattering detection (ELSD). However, high protein concentration can adversely impact method performance due to high sample viscosity, gel formation, column clogging, interfering peaks and loss of accuracy. To overcome this, a new method was developed based on EDTA mediated ethanol protein precipitation (EDTA/EtOH). This method was successfully implemented for the analysis of polysorbate in antibody formulations with wide range of protein concentration (10-250 mg/mL).

Enhancement of oleanolic acid concentration through acid hydrolysis of saponin-rich extracts from Chenopodium berlandieri.

The study investigated Chenopodium berlandieri to analyze its oleanolic acid (OA) content. Response surface methodology with central composite design was used to improve saponin extraction, varying temperature, ethanol, and sample-to-solvent ratio. Best conditions (65 °C, 50% ethanol, 1:10 ratio) yielded 53.45 ± 0.63 mg/g of extract from Huauzontle seeds. Temperature linearly impacted extract yield, while temperature and ethanol influenced total saponin content. Hydrolyzing saponin-rich extracts produced OA-rich extracts. Characterization via HPLC-ELSD and LC-MS identified OA4 as the most concentrated OA saponin (5.54 ± 0.16 mg/g). OA alone reached 2.02 ± 0.12 mg/g. Acid hydrolysis increased OA content by up to 3.27×, highlighting the potential of hydrolyzed Huauzontle extracts as a natural ingredient for various industries due to enhanced OA content.

Antioxidant Activity and Inhibition of Digestive Enzymes of New Strawberry Tree Fruit/Apple Smoothies.

In this study, original smoothies obtained with strawberry tree fruit puree and apple juice enriched with Diospyros kaki fruits, Myrtus communis purple berry extract, Acca sellowiana, and Crocus sativus petal juice were evaluated for their antioxidant activity and inhibition of targeted digestive enzymes. Values of CUPRAC, FRAP, ORAC, DPPH•, and ABTS•+ assays generally increased with plant enrichment, particularly for A. sellowiana addition (ABTS•+ 2.51 ± 0.01 mmol Trolox/100 g fw). The same trend was observed regarding the ability to scavenge reactive oxygen species (ROS) tested in Caco-2 cell cultures. Inhibitory activity on α-amylase and α-glucosidase was increased by D. kaki, M. communis, and A. sellowiana. Total polyphenols evaluated by UPLC-PDA analysis ranged between 535.75 ± 3.11 and 635.96 ± 5.21 mg/100 g fw, and A. sellowiana provided the higher amount. Flavan-3-ols accounted for more than 70% of phenolic compounds, and only smoothies enriched with C. sativus showed a high amount of anthocyanins (25.12 ± 0.18 mg/100 g fw). The outcome of this study indicates these original smoothies as a possible ally in counteracting oxidative stress, as established by their favourable antioxidant compound profile, thus suggesting an interesting future application as nutraceuticals.

[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.

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.

Identification and quantification of cyclitols and sugars isolated from different morphological parts of Raphanus sativus L.

Through this study, we aimed to develop a new analytical method for identification and quantification of sugars and cyclitols isolated from different morphological parts of Raphanus sativus L (R. sativus). Accelerated solvent extraction with water was involved for targets extraction. Solid phase extraction was used for purification and preconcentration, while high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD) was used for chromatographic analyses. A short method of only 30 min for a single analysis was developed finally. The obtained results, allowed for quantification of eight targets, i.e., three cyclitols (D-pinitol, allo-inositol and scyllo-inositol) and five sugars (xylose, D-mannose, D-fructose, D-glucose and sucrose) that were determined simultaneously using a single analysis. The developed method can be applied in industry as a routine method for analysis of sugars and cyclitols from different sources.

Quality evaluation methods of Poria based on yield and content of alkali extracted polysaccharide / 中国中药杂志

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 β-(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.

Discrimination of three varieties of Codonopsis Radix based on fingerprint profiles of oligosaccharides by high performance liquid chromatography- evaporative light scattering detector combined with multivariate analysis.

Codonopsis Radix (CR) is a plant that is important in the practice of traditional Chinese medicine (TCM). The Chinese Pharmacopoeia 2020 records dried roots prepared from three varieties of Campanulaceae plants under the designation CR ("Dang-shen" in Chinese), including Codonopsis pilosula (Franch.) Nannf (C. pilosula), Codonopsis pilosula Nannf.var.modesta (Nannf.) L81. T. Shen (C. pilosula var. modesta) and Codonopsis tangshen Oliv (C. tangshen). As major constituents of CR, oligosaccharides might contribute to its clinical efficacy except for other known active compounds, yet the differences in the oligosaccharide profiles of these three varieties of CR remain incompletely understood. In the present study, 135 samples from these different CR varieties were harvested, and oligosaccharide fingerprints for these samples were characterized via HPLC-ELSD, with 19 common peaks being matched. Oligosaccharides were further identified through the combination of electrospray ionization MS/MS (ESI-MS/MS) with nuclear magnetic resonance (NMR). Principal component analysis and linear discriminant analysis (LDA) approaches were then used to compare the oligosaccharide profiles of these three CR varieties. These analyses ultimately revealed that CR was compared with principal component analysis (PCA) and linear discriminant (LDA) methods. The analyses ultimately revealed that these CR samples contained high levels of inulin- and levan-type fructooligosaccharides (FOS), with variations in the relative levels of these FOS compounds among the three analyzed CR varieties. Through the combined analysis of oligosaccharide fingerprints and LDA results, it was possible to differentiate among these CR varieties, with an accurate classification rate of 96.3% and a cross-validation rate of 95.6%. Together, these results highlight a valuable approach to the classification and identification of different CR varieties.

Differentiation of three Asparagus species by UHPLC-MS/MS based molecular networking identification and chemical profile analysis.

Asparagi Radix (AR), a traditional Chinese medicine, is the dried roots of Asparagus cochinchinensis (Lour.) Merr. Modern pharmacological studies have shown that AR has various excellent bioactivities, such as antioxidative, antitumor, antibacterial, anti-inflammatory, and hypoglycemic effects. However, the quality control method of AR is incomplete and there are various AR adulterants in markets due to their similar morphological characters. Here, holistic and practical quality evaluation methods were developed to chemically distinguish three common Asparagus species in markets, including Asparagus cochinchinensis (Lour.) Merr., Asparagus officinalis L., and Asparagus lycopodineus (Baker) F.T.Wang & Tang. The chemical constituents of three species were rapidly tentatively annotated using a combination of ultra-high pressure liquid chromatography-linear ion trap-orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap-MS) and molecular networking (MN). Fifty-six steroidal saponins were annotated, including common and characteristic chemical constituents of the three Asparagus species. Besides, to establish holistic and practical methods to differentiate three Asparagus species, an HPLC-ELSD (evaporative light scattering detector) was applied for fingerprint analysis and content determination of the sum of protoneodioscin and protodioscin of twenty samples. Each Asparagus species showed characteristic chemical profile and AR showed much higher level of the sum of protoneodioscin and protodioscin than that in the others. The above analyses showed that the three Asparagus species mainly contain steroidal saponins and the developed HPLC-ELSD profile of saponin can be used to differentiate them. In conclusion, this study reveals the different chemical constituents of three Asparagus species and provides relatively feasible quality evaluation methods for them which are essential for the rational utilization of these Asparagus species.

Anti-skin-aging effects of tissue-cultured mountain-grown ginseng and quantitative HPLC/ELSD analysis of major ginsenosides.

Mountain-grown ginseng has free radical scavenging activity and suppresses inflammation. We evaluated the anti-skin-aging effects of tissue-cultured mountain-grown ginseng (TG) and its major ginsenosides. The effect of three extracts of TG and ginsenosides Rg1 (1), Rf (2), Rb1 (3), Re (4), and Rd (5) on the secretion of matrix metalloproteinase-1 (MMP-1) and collagen type I alpha 1 (COLIA1) was compared with that of tumor necrosis factor-alpha (TNF-α) stimulation of human dermal fibroblasts (HDFs), as determined via enzyme-linked immunosorbent assay. An analytical high-performance liquid chromatographic method with evaporative light-scattering detection (HPLC/ELSD) was developed for the simultaneous determination of the major ginsenosides in TG obtained via supercritical fluid CO2 or ethanol extraction. TG residues obtained via supercritical fluid CO2 extraction (TG1) and TG not subject to extraction (TG3) suppressed MMP-1 secretion in TNF-α-stimulated HDFs. Major ginsenoside content was higher in the TG1 than in residues extracted with ethanol (TG2) and TG3; ginsenoside Rg1 (1) content was the highest among all TG residues. Among them, ginsenosides Rg1 (1) and Re (4) suppressed MMP-1 in TNF-α-stimulated HDFs, whereas ginsenosides Rb1 (3) and Rd (5) increased COLIA1. In conclusion, TG and its active ginsenosides may have anti-skin-aging effects. Ginsenoside Rg1 (1) may also be beneficial in ameliorating skin damage. HPLC/ELSD can identify major ginsenosides and supercritical fluid CO2 extraction can be applied during health supplement or drug development.

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 58.88 µg/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.

Lactational Changes of Phospholipids Content and Composition in Chinese Breast Milk.

Phospholipids are pivotal polar lipids in human milk and essential for infants' growth and development, especially in the brain and cognitive development. Its content and composition are affected by multiple factors and there exist discrepancies in different studies. In this study, we determined five major phospholipids classes (phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidylcholine, and sphingomyelin) in 2270 human milk samples collected from 0 to 400 days postpartum in six regions of China. The high-performance liquid chromatography coupled with an evaporative light scattering detector (HPLC-ELSD) was performed to quantify the phospholipids. Total phospholipid median (IQR) content was in a range between 170.38 ± 96.52 mg/L to 195.69 ± 81.80 mg/L during lactation and was higher concentrated in colostrum milk and later stage of lactation (after 200 days postpartum) compared with that in the samples collected between 10 to 45 days postpartum. Variations in five major sub-class phospholipids content were also observed across lactation stages (phosphatidylethanolamine: 52.61 ± 29.05 to 59.95 ± 41.74 mg/L; phosphatidylinositol: 17.65 ± 10.68 to 20.38 ± 8.55 mg/L; phosphatidylserine: 15.98 ± 9.02 to 22.77 ± 11.17 mg/L; phosphatidylcholine: 34.13 ± 25.33 to 48.64 ± 19.73 mg/L; sphingomyelin: 41.35 ± 20.31 to 54.79 ± 35.26 mg/L). Phosphatidylethanolamine (29.18-32.52%), phosphatidylcholine (19.90-25.04%) and sphingomyelin (22.39-29.17%) were the dominant sub-class phospholipids in Chinese breast milk during the whole lactation period. These results updated phospholipids data in Chinese human milk and could provide evidence for better development of secure and effective human milk surrogates for infants without access to breast milk.

Effect of Apple Juice Enrichment with Selected Plant Materials: Focus on Bioactive Compounds and Antioxidant Activity.

Using a multi-analytical approach, this paper aimed to investigate the effect of apple juice enrichment with Arbutus unedo and Diospyros kaki fruits, Myrtus communis berry extract, Acca sellowiana, or Crocus sativus flower by-products on both bioactive compounds content and antioxidant activity. Physico-chemical parameters, vitamin C, sugars, organic acids, total polyphenol content, antioxidant activity, and sensory attributes were evaluated. An LC-PDA/MS QTof analysis allowed for the identification of 80 different phenolic compounds. The highest polyphenol content (179.84 and 194.06 mg of GAE/100 g fw) and antioxidant activity (CUPRAC, 6.01 and 7.04 mmol of Fe2+/100 g fw) were observed in products with added A. sellowiana and D. kaki, respectively. Furthermore, the study showed a positive correlation between polymeric procyanidins and antioxidant activity (0.7646-0.8539). The addition of A. unedo fruits had a positively significant influence on the increment of vitamin C (23.68 ± 0.23 mg/100 g fw). The obtained products were attractive to consumers, especially those with 0.1% C. sativus flower juice, M. communis berry extract, and persimmon D. kaki fruits. The synergy among the different analytical techniques allowed us to obtain a complete set of information, demonstrating that the new apple smoothies were enriched in both different beneficial molecules for human health and in antioxidant activity.

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.

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.

Rapid Identification of Common Secondary Metabolites of Medicinal Herbs Using High-Performance Liquid Chromatography with Evaporative Light Scattering Detector in Extracts.

The discovery and identification of novel natural products of medicinal importance in the herbal medicine industry becomes a challenge. The complexity of this process can be reduced by dereplication strategies. The current study includes a method based on high-performance liquid chromatography (HPLC), using the evaporative light scattering detector (ELSD) to identify the 12 most common secondary metabolites in plant extracts. Twelve compounds including rutin, taxifolin, quercetin, apigenin, kaempferol, betulinic acid, oleanolic acid, betulin, lupeol, stigmasterol, and ß-sitosterol were analyzed simultaneously. The polarity of the compounds varied greatly from highly polar (flavonoids) to non-polar (triterpenes and sterols). This method was also tested for HPLC-DAD and HPLC-ESI-MS/MS analysis. Oleanolic acid and ursolic acid could not be separated in HPLC-ELSD analysis but were differentiated using LC-ESI-MS/MS analysis due to different fragment ions. The regression values (R2 > 0.996) showed good linearity in the range of 50-1000 µg/mL for all compounds. The range of LOD and LOQ values were 7.76-38.30 µg/mL and 23.52-116.06 µg/mL, respectively. %RSD and % trueness values of inter and intraday studies were mostly <10%. This method was applied on 10 species of medicinal plants. The dereplication strategy has the potential to facilitate and shorten the identification process of common secondary metabolites in complex plant extracts.

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.

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 of alkanolamines in liquid cement grinding aids by HPLC coupled with evaporative light scattering detector.

Triethanolamine (TEA), triisopropanolamine (TIPA), diethanol isopropanolamine (DEIPA) are necessary cement additives, and knowing their contents is needed for quality control and also to understand final properties of the cement. Whether it is the production of grinding aids, technical research and development or application research all involve the detection of grinding aids. This work developed a simple analytical technique for the simultaneous analysis of these alkanolamines in liquid cement grinding aids using high-performance liquid chromatography (HPLC) combined with evaporative light scattering detection (ELSD). HPLC was conducted by an XBridge C18 column (with dimensions 4.6 × 250 mm and 5 µm particles) using methanol and 0.1% trichloroacetic acid as mobile elution phases. The ELSD sprayer and drift tube temperatures were 60 ºC and 90 ºC, respectively. HPLC-ELSD developed in this work demonstrated 1) high sensitivity with limits of detection for the three analytes are 0.15, 0.54, 1.04 µg/mL; 2) good linearity with correlation coefficients equal to 0.997-0.999 over the tested concentration range; 3) excellent repeatability with intra- and interday coefficient of variation (CV) below 2.71% and 4, satisfactory accuracy with recovery in the 95.5%-100.8% range. This novel method is a powerful, time- and costeffective tool for alkanolamine analyses and quality control.

Application of ATR-FTIR spectroscopy for profiling of non-structural carbohydrates in onion (Allium cepa L.) bulbs.

Qualitative and quantitative composition of non-structural carbohydrates comprising glucose, fructose, sucrose and fructooligosaccharides (FOS) is one of the key determinants of market suitability, storability and technological processability of onions. To develop a cost-effective and rapid tool for carbohydrate profiling, applicability of attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy of onion juice was investigated with special regard to FOS patterns. As reference, detailed carbohydrate profiles of onion juices were generated by high-performance liquid chromatography coupled with evaporative light scattering detection (HPLC-ELSD). Hierarchical cluster analysis (HCA) of ATR-FTIR spectra was successfully applied for classifying onions into fresh market, storage and dehydrator type according to HPLC-ELSD profiles. A bootstrapping method for automatized test-set validation by projection to latent structures (PLS) algorithms using HPLC and ATR-FTIR spectroscopy data was developed. Model statistics showed promising perspectives for reliable quantification of individual saccharides and sum parameters. The presented methodology allows estimating the nutritional and pre-biotic value directly during cultivation and processing.