Normal-phase High-performance Liquid Chromatography Research Articles

Estimation of Diafenthiuron Residues in Cardamom (Elettaria cardamomum (L.) Maton) Using Normal Phase HPLC: Dissipation Pattern and Safe Waiting Period in Green and Cured Cardamom Capsules

Diafenthiuron is an effective insecticide used for pest management in cardamom. Residues of diafenthiuron and its degradation/dissipation pattern in cardamom were determined to work out safe waiting period. Samples were collected after three sprays of diafenthiuron @ 400 and 800 g a.i ha−1 and the residues extracted in acetonitrile and quantified in normal phase HPLC in UV detector. Diafenthiuron was detected in 6.61±0.1 min. The limits of detection (LOD) and limits of quantification (LOQ) were determined to be 0.01 and 0.05 μgmL−1. The initial deposits were found to be 3.82 and 4.10 μg g−1 after sprays of diafenthiuron @ 400 g a.i ha−1 in the first and second experiments, respectively. Nearly cent percent of residues dissipated at 10 days after treatment in the recommended dose of diafenthiuron 400 g a.i ha−1 and the half life varied from 2.0 to 2.8 days with a waiting period of 5.5 to 6.7 days in green capsules of cardamom. The waiting period was 5.4 to 7.0 days in cured capsules of cardamom. With harvest being the focal point for enforcement of residue tolerances, the suggested waiting period of seven days is safe without the problem of pesticide residues in harvestable produce.

Enantioselective Degradation of Dufulin in Four Types of Soil

In this study, enantioselective degradation of dufulin in four types of soil (Guiyang silty loam, Nanning silty clay, Hefei silty clay, and Harbin silty clay) was investigated under sterile and nonsterile conditions. Pesticide residues in soil samples were extracted with acetonitrile. S-(+)-Dufulin and R-(-)-dufulin were separated and determined on an amylose tris(3,5-dimethylphenylcarbamate) (Chiralpak IA) chiral column by normal phase high-performance liquid chromatography (HPLC). The absolute configurations of dufulin enantiomers were determined by obtaining experimental and computed circular dichroism spectra. Dufulin enantiomers were found to be configurationally stable in the selected soils, and no interconversion was observed during the incubation of enantiopure S-(+)- or R-(-)-dufulin under nonsterile conditions. Compared to the half-life (t1/2) of dufulin in sterile soils, the degradation rate was higher in nonsterile soils, which suggests that dufulin degradation can be attributed primarily to microbial activity in soils used for agricultural cultivation. Furthermore, enantiopure S-(+)-dufulin degraded more rapidly than its antipode. This suggests that use of enantiopure S-(+)-dufulin could exert less disturbance to soil bioactivity and contribute less to environmental pollution.

A Novel Method for the Synthesis of Latanoprost

Latanoprost is a kind of PGF2α analogues, which is now one of the first-choice drugs in the clinic for open angle glaucoma and ocular hypertension. However, the previous synthetic methods have problems of long steps, low yield and diffi- cult separation of isomeric impurities. Herein, we report a novel method for the synthesis of latanoprost in ten steps. Benzoyl Corey lactone as starting material was oxidized to form corresponding aldehyde by Dess-Martin oxidation. The ω side chain was bonded by improving Horner-Wadsworth-Emmons reaction with dimethyl(2-oxo-4-phenylbutyl)phosphonate and lithium chloride, and reduced with (-)-diisopinocampheyl chloroborane at -30 ℃ because of its greater selectivity towards the production of desired S-isomer (95%). The deprotection step wherein the protecting group of benzoyl on the hydroxyl group of the cyclopentane ring was removed, was preferably carried out by potassium carbonate in methanol. The hydrogenation of double bond in ω side chain was carried out by using 5% palladium-carbon as catalyst. The tetrahydropyranyl (THP) group was used for the protection of the diol with p-toluenesulfonic acid as catalyst. Latanoprost lactol was obtained by reducing the lactone with diisobutylaluminum hydride (DIBAL) at -78 ℃. α side chain of latanoprost was bonded by Wittig reaction with 4-carboxybutyl triphenylphosphoium bromide and NaHMDS forming phosphorus ylide. The carboxylic acid was alky- lated with isopropyl iodide in the presence of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU), and then removed the THP protect- ing groups with pyridinium 4-toluenesulfonate (PPTS). Latanoprost was separated from two isomers of 15(S)-latanoprost and 5,6-trans-latanoprost after purification by normal-phase high performance liquid chromatography. Thus latanoprost was ob- tained with high purity of 99.91% and high overall yield of 19.2%, and the structure was characterized by 1 H NMR, 13 C NMR, IR and HRMS. Compared with the previous routes, the current synthesis is shorter, more practical, and more suitable for large-scale preparation.

The development and validation of HPLC method for quantification of DL-α-tocopherol in quinoa seeds (Chenopodium quinoa willd.)

Aiming to achieve a precise method with easy application, this study presents the development and validation of a fast method for quantification of vitamin DL-α-tocopherol in quinoa seeds. The methodology was based on an extraction procedure using ultrasonic bath and determination by normal-phase HPLC with UV-VIS detector. Validation parameters showed adequate linearity, relative standard deviations between 0.5 and 0.8% (n=10), limits of detection and quantification were 3.0 ng/mL and 11.0 ng/mL, respectively. Moreover, testing the robustness of the method suggested that it was not changeable with time or condition. The results showed that this method is accurate and simple and thus applicable in laboratories for determination of DL-α-tocopherol in quinoa seeds.

Determination of (R)-timolol in (S)-timolol maleate active pharmaceutical ingredient: Validation of a new supercritical fluid chromatography method with an established normal phase liquid chromatography method

An enantioselective supercritical fluid chromatography (SFC) method was developed and validated to meet the current European Pharmacopoeia requirements of a limit test for the determination of S-timolol maleate enantiomeric purity in timolol maleate drug substance. The developed method is presented as an alternative to the current normal phase high performance liquid chromatography (NP-HPLC) method described in the European Pharmacopoeia (Timolol Maleate Monograph). Using a 4.6mm×250mm Chiralcel OD-H (dp: 5μm) column and a mobile phase of (93:7) CO2/0.1% (v/v) TEA in MeOH delivered at 4.0mLmin−1 resolution of 2.0 was achieved within 5min, representing a 3-fold reduction in run-time and an 11-fold reduction in solvent consumption relative to the NP-HPLC method. Method robustness was examined by the variation of flow rate (±0.5mLmin−1), column temperature (±5°C) and column back-pressure (±10bar) and resolution was maintained at ≥1.9 in all cases. R-timolol was resolved from all potential impurities and the limit of detection was improved by increasing the sample concentration threefold compared to the NP-HPLC method such that the method could detect the R-timolol enantiomer at 0.5% (w/w) with respect to S-timolol maleate. Additional validation parameters demonstrated that the potential of the method to be used for routine release testing of timolol maleate raw material for drug product manufacturing in which the quantitation of R-timolol impurity in S-timolol maleate drug substance would be a requirement.

Response to Bernstein et al

Sir, We welcome the letter by Bernstein et al1 in response to our publication ‘What is meso-zeaxanthin, and where does it come from?' in Eye 2013.2 In their letter, Bernstein and colleagues argue that our review article contains ‘several critical errors that need to be considered.' Bernstein and colleagues endeavour to make their points under the following headings: Quantitation of xanthophylls using reverse- and normal-phase HPLC. The role of saponification in the quantitation of xanthophylls in food and supplements. Meso-zeaxanthin in lutein supplements. Additional evidence supporting lutein as the precursor of meso-zeaxanthin. In our letter below, we reply directly to these points in normal font. Statements made by Bernstein and colleagues are presented in bold font for clarity.

Tocopherol profile of Brazil nut oil from different geographic areas of the Amazon region

The tocopherol content of Brazil nut oil from different Amazon regions (Manicoré-AM, Rio Preto da Eva-AM, São João da Baliza-RR, Caroebe-RR, Belém-PA, and Xapurí-AC) was investigated by normal-phase high-performance liquid chromatography. For all authentic oils, two isomers: α- and γ-tocopherols were observed (37.92-74.48 µg g-1, 106.88-171.80 µg g-1, respectively), and their levels were relatively constant among the oils having these geographic origins, which would enable to distinguish Brazil nut oil from other plant oils for authentication purposes. Commercial Brazil nut oils were also evaluated, and some of these oils demonstrated a tocopherol content that was very different from that of the authentic oils. Therefore, we suggest that the tocopherol profile of Brazil nut oil can be useful chemical marker for quality control and authentication.

Phytochemicals in fruits of Hawaiian wild cranberry relatives

Cranberries (Vaccinium macrocarpon Ait.) contain high levels of phytochemicals such as proanthocyanidins (PACs). These polymeric condensations of flavan-3-ol monomers are associated with health benefits. Our objective was to evaluate phytochemicals in fruit from Hawaiian cranberry relatives, V. reticulatum Sm. and V. calycinum Sm. Normal-phase HPLC coupled with fluorescence and ESI-MS detected PACs; the colorimetric 4-dimethylaminocinnamaldehyde (DMAC) assay was used to determine total PACs. Spectrophotometric tests and reverse-phase HPLC coupled to photodiode array and refractive index detectors evaluated phenolics, sugars, and organic acids. Antioxidant capacity was determined by the ORAC and FRAP assays. Antioxidant capacities of Hawaiian berries were high. The FRAP measurement for V. calycinum was 454.7 ± 90.2 µmol L(-1) Trolox equivalents kg(-1) for pressed fruit. Hawaiian berries had lower peonidin, quinic and citric acids amounts and invert (∼1) glucose/fructose ratio compared with cranberry. Both Hawaiian Vaccinium species were good sources of PACs; they contained phenolics and PAC monomers, A and B-type trimers, tetramers and larger polymers. Vaccinium reticulatum and V. calycinum showed comparable or higher PAC levels than in cranberry. Cranberries had higher percentage of A-type dimers than did V. reticulatum. A and B-type dimers were not differentiated in V. calycinum. The total PACs (as measured by DMAC) for V. calycinum (24.3 ± 0.10 mg catechin equivalents kg(-1) ) were about twice that in cranberry. Berries of V. reticulatum and V. calycinum could serve as a rich dietary source of PACs, comparable to or greater than cranberries. These finding suggest that Hawaiian Vaccinium berries could be a functional food. Additional examination of the phytochemicals in other wild Vaccinium species is warranted.

Validated HPLC Method and Temperature Stabilities for Oil-Soluble Organosulfur Compounds in Garlic Macerated Oil

To enhance the utilization of garlic macerated oil as functional foods, oil-soluble organosulfur compounds were investigated using normal-phase high-performance liquid chromatography method. For analysis of compounds, it was simply extracted with 98% n-hexane in 2-propanol followed by sensitive and selective determination of all compounds. These method exhibited excellent linearity for oil-soluble organosulfur compounds with good coefficient (r > 0.999). Average recoveries were in the range of 80.23-106.18%. The limits of quantitation of oil-soluble organosulfur compounds ranged from 0.32 to 9.56 μg mL(-1) and the limits of detection were from 0.11 to 3.16 μg mL(-1). Overall, the precision of the results, expressed as relative standard deviation, ranged from 0.55 to 11.67%. The proposed method was applied to determining the contents of oil-soluble organosulfur compounds in commercial garlic macerated oils. Also, the stability of oil-soluble organosulfur compounds in garlic macerated oil were evaluated during 3 months of storage at four difference temperatures (4, 10, 25 and 35°C). The results showed the studied oil-soluble compounds in garlic macerated oil were stable at 4°C and relatively unstable at 35°C with varied extents degradation. Therefore, these validation data and temperature stability may be useful for quality evaluation of garlic macerated oils.

Characterization of Phospholipid Molecular Species and Peptide Molecules in Wheat Sprout Hydroalcoholic Extract

The phospholipid molecular species and the main peptide molecules of wheat sprout hydroalcoholic extract have been fully characterized by normal-phase high performance liquid chromatography coupled online with positive electrospray ionization tandem mass spectrometry. The extract that resulted was rich in phospholipid molecular species formed by the combination of the two essential fatty acids (α-linoleic and α-linolenic). These species accounted for 51.7% of total phosphatidic acid, 47.3% of total phosphatidylethanolamine, 37.7% of total phosphatidylcholine, and 14.1% of total phosphatidylinositol. The last one showed the highest amounts of species containing palmitic acid, thus representing the most saturated phospholipid class. The extract was also shown to contain several peptide sequences with both potential antioxidant domains and interaction sites for phospholipids (i.e., H-Ala-Gly-Ser-Met-Met-Cys-NH2, H-Tyr-Met-Thr-Val-Val-Ala-Cys-NH2, etc.); this latter finding can have a highly positive impact on the poor peptides bioavailability. Because of the presence of essential fatty acids-rich phospholipids and bioactive peptides, wheat sprout hydroalcoholic extract can be considered a potential functional food ingredient.

Association of HDL-related loci with age-related macular degeneration and plasma lutein and zeaxanthin: the Alienor study.

BackgroundSeveral genes implicated in high-density lipoprotein (HDL) metabolism have been reported to be associated with age-related macular degeneration (AMD). Furthermore, HDL transport the two carotenoids, lutein and zeaxanthin, which are highly suspected to play a key-role in the protection against AMD. The objective is to confirm the associations of HDL-related loci with AMD and to assess their associations with plasma lutein and zeaxanthin concentrations.MethodsAlienor study is a prospective population-based study on nutrition and age-related eye diseases performed in 963 elderly residents of Bordeaux, France. AMD was graded according to the international classification, from non-mydriatic colour retinal photographs. Plasma lutein and zeaxanthin were determined by normal-phase high-performance liquid chromatography. The following polymorphisms were studied: rs493258 and rs10468017 (LIPC), rs3764261 (CETP), rs12678919 (LPL) and rs1883025 (ABCA1).ResultsAfter multivariate adjustment, the TT genotype of the LIPC rs493258 variant was significantly associated with a reduced risk for early and late AMD (OR=0.64, 95%CI: 0.41-0.99; p=0.049 and OR=0.26, 95%CI: 0.08-0.85; p=0.03, respectively), and with higher plasma zeaxanthin concentrations (p=0.03), while plasma lipids were not significantly different according to this SNP. Besides, the LPL variant was associated with early AMD (OR=0.67, 95%CI: 0.45-1.00; p=0.05) and both with plasma lipids and plasma lutein (p=0.047). Associations of LIPC rs10468017, CETP and ABCA1 polymorphisms with AMD did not reach statistical significance.ConclusionThese findings suggest that LIPC and LPL genes could both modify the risk for AMD and the metabolism of lutein and zeaxanthin.

Determination of eight isomers and related substance of Aprepitant using normal-phase and reverse-phase HPLC methods with mass spectrophotometric detection

Introduction: Aprepitant is a chiral prodrug possessing three stereogenic centers and exists in eight stereochemical forms. It was aimed to estimate and validate Aprepitant using reverse phase HPLC. Materials and methods: Chiral liquid chromatography with an ion-trap mass spectrophotometer was used to isolate all the isomers. The separation of the eight isomers was achieved on an amylase-based chiral column (Chiralpak ADH, 250 mm � 4.6 mm) using n-hexane/isopropyl alcohol/methanol/trifluoroacetic acid (970/40/4/0.5, vol/vol/vol/vol) as the mobile phase at a flow rate of 0.5 ml/min. A reverse-phase HPLC method was used to analyze Aprepitant and its related substances. The determination of Aprepitant and its related substances was developed by using Xterra RP e 18, 250 mm � 4.6 mm, 5 mm column. Results and conclusion: These methods were validated according to the International Conference on Harmonization (ICH) guidelines. The limit of detection (LOD) and limit of quantitation (LOQ) for Aprepitant were found to be 0.14 mg/ml and 0.41 mg/ml, respectively, for chiral substance and 0.07 mg/ml and 0.21 mg/ml, respectively, for the related substance.

Direct Coupling of Normal-Phase High-Performance Liquid Chromatography to Atmospheric Pressure Laser Ionization Fourier Transform Ion Cyclotron Resonance Mass Spectrometry for the Characterization of Crude Oil

The high complexity of crude oil makes the use of chromatographic separation an important tool especially for sample simplification. The coupling of normal-phase high-performance liquid chromatography (HPLC) using a polar aminocyano column to a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer offers the best attributes of good separation prior to ultrahigh resolution mass spectrometry (MS) detection. Atmospheric pressure laser ionization (APLI) was used as an ionization technique to analyze the nitrogen-containing aromatic compounds in a deasphalted crude oil due to its unique selectivity toward aromatic compounds and also due to its sensitivity. Two main chromatographic peaks were observed during this separation indicating a class-based separation. Mass spectra obtained from fractions were collected along the entire retention time and compared with each other to assign the unique constituents. By coupling the HPLC system directly to the FTICR mass spectrometer, comparable ion and UV chromatograms were obtained, reflecting the scan-to-scan sensitivity of the coupling system. The results show that it is possible to calculate reconstructed class chromatograms (RCC), allowing differences in class composition to be traced along the retention time. As an example, radical and protonated nitrogen species generated by APLI were detected along the retention time which enabled a differentiation between basic and nonbasic species in the same polar peak, thus overcoming the limitation of chromatographic resolution. This report represents the first online LC-FTICR MS coupling in the field of crude oil analysis.

Preparation of Bonded Cellulose Tris(3,5-dimethylphenylcarbamate) Chiral Stationary Phases by Using Three Bifunctional Reagents

Three di-acyl chlolide reagents, adipoyl chloride, terephthaloyl chloride and isophthaloyl chloride, were used as spacer reagents to prepare bonded type of three cellulose (3,5-dimethylphenyl)carbamate (CDMPC) chiral stationary phases (CSPs). The CDMPC CSPs were prepared using these three acid chlorides as spacer agents at the 6-position of the primary hydroxyl group on the glucose unit of cellulose regioselectively. The chiral recognition ability of the prepared CSPs for five racemates was evaluated by normal-phase high-performance liquid chromatography (HPLC) with the following mobile phases: hexane/isopropanol (IPA), hexane/IPA/ tetrahydrofuran (THF) and hexane/IPA/chloroform. The result showed that these prepared CSPs can be used in THF and chloroform solutions and the chiral recognition abilities of the CSPs were improved depending on the eluents and chiral samples.

Analysis of the Structure and Stability of Erythropoietin by pH and Temperature Changes using Various LC/MS

The purpose of stability testing is to provide evidence about how the quality of a drug varies with time under the influence of a variety of environmental factors. In this study, erythropoietin (EPO) was analyzed under different pH (pH 3 and pH 9) and temperature (<TEX>$25^{\circ}C$</TEX> and <TEX>$40^{\circ}C$</TEX>) conditions according to current Good Manufacturing Practice (cGMP) and International Conference on Harmonisation (ICH) guidelines. The molecular weight difference between intact EPO and deglycosylated EPO was determined by SDS-PAGE, and aggregated forms of EPO under thermal stress and high-pH conditions were investigated by size exclusion chromatography. High pH and high temperature induced increases in dimer and high molecular weight aggregate forms of EPO. UPLC-ESI-TOF-MS was applied to analyze the changed modification sites on EPO. Further, normal-phase high-performance liquid chromatography was performed to identify proposed glycan structures and high pH anion exchange chromatography was carried out to investigate any change in carbohydrate composition. The results demonstrated that there were no changes in modification sites or the glycan structure under severe conditions; however, the number of dimers and aggregates increased at <TEX>$40^{\circ}C$</TEX> and pH 9, respectively.

Increased bisecting N‐acetylglucosamine and decreased branched chain glycans of N‐linked glycoproteins in expressed prostatic secretions associated with prostate cancer progression

Using prostatic fluids rich in glycoproteins like prostate-specific antigen and prostatic acid phosphatase (PAP), the goal of this study was to identify the structural types and relative abundance of glycans associated with prostate cancer status for subsequent use in emerging MS-based glycopeptide analysis platforms. A series of pooled samples of expressed prostatic secretions (EPS) and exosomes reflecting different stages of prostate cancer disease were used for N-linked glycan profiling by three complementary methods, MALDI-TOF profiling, normal-phase HPLC separation, and triple quadropole MS analysis of PAP glycopeptides. Glycan profiling of N-linked glycans from different EPS fluids indicated a global decrease in larger branched tri- and tetra-antennary glycans. Differential exoglycosidase treatments indicated a substantial increase in bisecting N-acetylglucosamines correlated with disease severity. A triple quadrupole MS analysis of the N-linked glycopeptides sites from PAP in aggressive prostate cancer pools was done to cross-reference with the glycan profiling data. Changes in glycosylation as detected in EPS fluids reflect the clinical status of prostate cancer. Defining these molecular signatures at the glycopeptide level in individual samples could improve current approaches of diagnosis and prognosis.

Identification of antioxidant components and fatty acid profiles of the leaves and fruits from Averrhoa carambola

The antioxidant compounds of leaves and fruits from Averrhoa carambola were extracted with acetone:water (7:3, v/v) and distilled water. The acetone:water extracts (AWE) were separated into five different subfractions, three obtained by liquid–liquid extraction using n-hexane (HF), ethyl acetate (EAF) and water (WF), and the other two obtained by further fractionation of the WF on a Sephadex LH-20 column (WS1 and WS2). The antioxidant activities, total phenolics and total flavonoids of these extracts and fractions were investigated. Results showed that all the extracts and fractions possessed potent antioxidant activities, as measured by DPPH, ABTS and FRAP methods, with the strongest for WS2 fractions accompanied with the highest total phenolics and total flavonoids. Reversed-phase HPLC, normal-phase HPLC, MALDI-TOF-MS and 13C NMR analyses identified that the main antioxidant components present in WS2 fractions were essentially of procyanidin-type proanthocyanidins, consisting mainly of epicatechin units linked by B-type interflavan bonds. In addition, GC–MS analysis revealed that the most abundant fatty acids were α-linolenic acid (62.04 ± 1.65%) for leaves and oleic acid (55.44 ± 1.37%) for fruits. The amount of total unsaturated fatty acids in leaves and fruits comprised more than 77% of total fatty acid.

Simultaneous Determination of 13-cis and all-trans Vitamin A Palmitate (Retinyl Palmitate), Vitamin A Acetate (Retinyl Acetate), and Total Vitamin E (α-Tocopherol and DL-α-Tocopherol Acetate) in Infant Formula and Adult Nutritionals by Normal Phase HPLC: First Action 2012.10

This HPLC method, with both variable UV and fluorescence detection, allows for the simultaneous determination of vitamin A palmitate, vitamin A acetate, and total vitamin E in infant, pediatric, and adult nutritional formulas. The concentration of each vitamin form is calculated by comparison with standards of known concentration. Following hydrolysis, the vitamins are extracted into iso-octane and analyzed by normal phase (NP) HPLC. The method was evaluated for linearity, precision, and accuracy using a selection of the Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) matrixes, including milk-based, soy-based, and hydrolyzed protein, as well as high- and low-fat products. A single-laboratory validation has been completed for all analytes using a selection of SPIFAN matrixes. Performance parameters included a working range of 2-450 microg/100 g ready-to-feed for vitamin A and 0.03-8.0 mgl100 g reconstituted final product for vitamin E. LOD was <1.0 microg and <0.1 mgl100 g reconstituted final product for vitamins A and E, respectively; RSD was 1.08-8.70% over a range of concentration; and average recoveries of 97.4-101.3%. Repeatability of <4% for vitamin A and <8% for vitamin E was calculated from five laboratories using this method. Results indicate that this method is suitable for the analysis of vitamins A and E in all forms of infant, adult, and pediatric formulas (powders, ready-to-feed liquids, and liquid concentrates). The Expert Review Panel (ERP) of Infant Formula reviewed this method separately for vitamins A and E, including all available method validation data at the AOAC INTERNATIONAL Annual Meeting on September 29, 2012. Following evaluation of the data for both methods, the ERP agreed that both methods met the standard method performance requirements articulated by SPIFAN. The ERP granted First Action status to both methods, and recommended that a single method be published for the simultaneous determination of vitamin A palmitate, vitamin A acetate, and total vitamin E (DL-a-tocopherol and DL-alpha-tocopherol acetate) in infant formula and adult nutritionals by NP HPLC.

Underutilized Chokeberry (Aronia melanocarpa, Aronia arbutifolia, Aronia prunifolia) Accessions Are Rich Sources of Anthocyanins, Flavonoids, Hydroxycinnamic Acids, and Proanthocyanidins

Polyphenols from underutilized black, purple, and red aronia (Aronia melanocarpa, Aronia prunifolia, and Aronia arbutifolia) and 'Viking' (Aronia mitschurinii) berries were characterized. Anthocyanin and nonanthocyanin flavonoids were quantitated by UHPLC-DAD-MS and proanthocyanidins by normal-phase HPLC. On a dry weight basis, anthocyanins were mainly cyanidin-3-galactoside, highest in black aronia (3.4-14.8 mg/g) and lowest in red aronia (0.5-0.8 mg/g) as cyandin-3-galactoside equivalents. Berries from 'Viking' and the red accession UC021 had substantially more proanthocyanidins than the other accessions, with 3.3 and 3.8 mg catechin equiv/g, respectively. Chlorogenic acids and quercetin glycosides were most abundant in purple UC047 berries, at 17.3 and 1.3 mg/g, respectively. In contrast to anthocyanin content, total phenol values were highest in berries from red and purple accessions and attributed to phenolic acid and proanthocyanin content. Thus, red, purple, and black aronia berries are rich sources of polyphenols with various levels of polyphenol classes.

Enhanced glycan nanoprofiling by weak anion exchange preparative chromatography, mild acid desialylation, and nanoliquid chromatography‐mass spectrometry with nanofluorescence detection

The structural characterization and quantification of the glycome of cells and glycoproteins is necessary for the understanding of glycan functions in Biology, the development of diagnostics tests, and the monitoring of glycoprotein pharmaceuticals. Classical N-glycan characterization methods involve enzymatic release followed by derivatization with a fluorochrome and separation by normal-phase HPLC. We have recently developed glycan nanoprofiling, a method for the simultaneous quantification and characterization of the N-glycans without the need of external standardization. Although glycan nanoprofiling allows the characterization of both neutral and sialylated glycans within the same chromatographic run, a significant drawback is the coelution of similar glycans when complex glycan mixtures are analyzed. To overcome this problem, we have developed enhanced glycan nanoprofiling. This new method introduces a weak anion-exchange HPLC separation step to fractionate glycans according to their sialic acid content followed by a mild acid desialylation. Glycans are then resolved by nano-LC-coupled ESI-MS with an intercalated nanofluorescence detector. Neutral glycans have a better analytical separation, better ionization profiles, and provide significantly higher MS signals allowing a detailed characterization of rare glycan species. Enhanced glycan nanoprofiling is a powerful approach that provides a fast and sensitive alternative to available N-glycan profiling methods.