Reversed-phase Column Chromatography Research Articles

Sintesis Tetrapeptida Linear (DPAP) Menggunakan Metode Sintesis Peptida Fasa Padat (SPPS) Dan Aktivitas Insektisidanya Terhadap Ulat Krop Kubis

Crop cabbage worm (Crocidolomia pavonana) is one of the pests that has a bad effect on the cabbage production in Indonesia. One of the efforts that has been applied to control this pest is by using bayrusil. However, the bayrusil can cause negative effects such as poisoning and environmentally polluting effects. To prevent the undesired effects, it has been developed the use of environmentally friendly insecticide and one of them is the use of insecticidal peptide. Tripsin-modulating oostatic factor (TMOF) and ist analogues is one of the peptides that has an insecticidal activity. The aim of this research is to synthesise a linear tetrapeptide (DPAP) an analogues of TMOF and to test insecticidal activities against crop cabbage worm. A DPAP has been synthesised by Solid Phase Peptide Synthesis on 2-chlorotrytil chloride resin. Fmoc strategy was applied on the synthesis and a combination DIC/Oxime was employed in the coupling reaction.The tetrapeptidyl resin was cleaved by TFA:water:EDT (90:5:5). The peptide was purified by reverse-phase column chromatography and characterized by TOF ES-MS spectroscopy. A linear peptide was biologically tested towards the C. pavonana, showing percentage of mortality values at 1000 ppm.

Toxicity of primulic acid 1 against a daphnid species Simocephalus expinosus s.l.

Abstract Primulic acid 1 is the main saponin present in Primula elatior. The present study describes the isolation of this amphiphilic compound from primula root. It was performed by ultrasonic maceration, reverse-phase column chromatography and crystallization. Investigations of its physicochemical property are represented by the determination of critical micelle concentration (cmc). The cmc value of the amphiphile was 9.4 × 10−5 mol·dm−3. The evaluation of environmental toxicity was performed on a daphnid species Simpocephalus expinosus s.l., which was very sensitive to primulic acids 1. The results from acute immobilisation test show that the tested compound has half maximal effective concentration after 24 hours (EC50-24 h) equal to 6.9 mg·l−1. Saponin can be classified as a toxic compound for aquatic organisms.

Quantitative structure–chromatographic retention relationship of synthesized peptides (HGRFG, NPNPT) and their derivatives

Carapax Trionycis extract peptides (HGRFG, NPNPT) are able to protect against CCl4-induced liver fibrosis. Therefore, this study applies to deal with chromatographic lipophilicity determination of synthesized peptides (HGRFG, NPNPT) and their derivatives using reversed-phase high performance liquid chromatography (RP-HPLC) combined with methanol-water mobile phase and two reversed-phase chromatographic columns (COSMOISL 5C18-MS-II and SHIMADZU-C18). The chromatographic lipophilicity of the analyzed compounds was expressed as logkw constant and correlated with lipophilicity descriptors. Quantitative structure-retention relationships (QSRR) analysis was performed to imitate chromatographic lipophilicity behavior using molecular descriptors. Modeling was performed using linear regression (LR) and multiple linear regression (MLR) methods with the help of principal component analysis (PCA) and hierarchical cluster analysis (HCA). The most influential molecular descriptors were lipophilicity descriptors, which are important for molecules ability to pass through biological membranes. All established QSRR models were statistically validated by standards, cross- and external validation parameters. According to these statistical validation parameters, MLR models (R2 > 0.856) were better for chromatographic lipophilicity prediction of peptide compounds. It can be concluded that chromatographic systems with COSMOISL 5C18-MS-II column were better for modeling of logkw than systems with SHIMADZU-C18 column. Modeling was performed in order to obtain lipophilicity profiles of investigated compounds as future drug candidates.

Integrated Strategy for Large-Scale Investigation on Protein Core Fucosylation Stoichiometry Based on Glycan-Simplification and Paired-Peaks-Extraction.

Core fucosylation (CF) is a special form of N-glycosylation and plays an important role in pathological and biological processes. Increasing efforts in this area are focused on the identification of CF glycosites, whereas evidence showed that the stoichiometry of CF occupancy is functionally important. Here, an integrated strategy based on "Glycan-Simplification and Paired-Peaks-extraction" (GSPPE) for detecting large-scale stoichiometries of CF was developed. After HILIC enrichment of intact glycopeptides, sequential cleavages by endoglycosidases H and endoglycosidases F3 were performed to generate simplified glycopeptide forms (SGFs), i.e., peptide-GlcNAc (pep-HN) and peptide-GlcNAc-Fucose (pep-CF). These paired SGFs were found to be eluted consecutively on a reversed-phase chromatography column, which allowed us to obtain peak areas of SGF pairs, even if only one of the peaks was captured by the mass spectrometer (MS), by introducing the Paired-Peaks-Extraction algorithm. Thus, the missing value dilemma of random data-dependent MS/MS acquisition was reduced and the stoichiometry of site-specific CF could be calculated. We systematically evaluated the feasibility of this strategy using standard glycoproteins and then explored urinary samples from healthy individuals and hepatocellular carcinoma (HCC) patients. In total, 1449 highly reliable core fucose glycosites and their corresponding CF stoichiometries were obtained. Dozens of glycosites that differed significantly in the urine of healthy individuals and HCC patients were disclosed. The developed approach and program presented here may promote studies on core fucosylation and lead to a deeper understanding of their dysregulation in physiological- or pathological processes.

Separation and Purification of Lipase Inhibitory Peptide from Fermented Milk by Lactobacillus plantarum Q180

In this study, we separated and purified lipase inhibitory peptide from fermented milk by Lactobacillus plantarum Q180 with the aim of developing a new functional anti-lipase activity yogurt product. L. plantarum 180 was inoculated into 10% reconstituted skimmed milk and incubated at 37°C until the pH of the culture reached pH 4.4. The lipase activity was measured using porcine pancreatic lipase. The lipase inhibitory peptides were gradually isolated by ultrafiltration, reversed phase column chromatography (RPC), reversed phase high-performance liquid chromatography (RP-HPLC), and gel permeation high-performance liquid chromatography (GP-HPLC) from the fermented milk by L. plantarum Q180. An ODS-AQ column was used for the RPC, a Vydac C18 column for the RP-HPLC, and a Superdex Peptide HR column for the GP-HPLC. The peptide was composed of Asp, Thr, Ile, Ser, Ala, and Gln, and the anti-lipase activity (IC50) was 2,817 μg/mL.

Enhanced Cordycepin Production in Caterpillar Medicinal Mushroom, Cordyceps militaris (Ascomycetes), Mutated by a Multifunctional Plasma Mutagenesis System.

A multifunctional plasma mutation system (MPMS) method was used to create high cordycepin-yielding mutations from wild Cordyceps militaris, which yielded many viable mutants, many of which produced more cordycepin compared to the wild strain. One particular mutant strain (GYS60) produced 7.883 mg/mL, which is much higher than those reported to date and is more than 20 times higher than that of the wild strain, whereas the cordycepin production of another viable mutant (GYS80) was almost zero. The extraction and purification of cordycepin, using the fermentation broth of C. militaris GYS60, was also investigated. Cordycepin was extracted by using AB-8 macroporous resin and purified by using reversed-phase column chromatography. When the sample was adsorbed onto the macroporous resin, 20% ethanol was used as the desorption solvent yielding various fractions. The fractions containing cordycepin were loaded onto a reversed-phase chromatography column packed with octadecyl bonded silica as the stationary phase and ethanol (95%)/acetic acid solution (5%) at pH 6.0 as the mobile phase. The combination of this two-step extraction-purification process yielded cordycepin at 95% purity with a total recovery rate of 90%.

Separation and indirect ultraviolet detection of piperidinium cations by using imidazolium ionic liquids in liquid chromatography

A method for the separation and indirect ultraviolet detection of piperidinium cations by using imidazolium ionic liquids in liquid chromatography was developed. This method uses the most common ultraviolet detector and C18 reversed-phase chromatographic column. The influences of imidazolium ionic liquids, ion-pair reagents, organic solvents, etc. on the separation and detection of piperidinium cations were investigated. The imidazolium ionic liquids in mobile phase are both the background ultraviolet absorption reagent of indirect ultraviolet detection and the active components to improve the separation of piperidinium cations. Compared with reversed-phase chromatography, reversed-phase ion-pair chromatography is a better method for the determination of piperidinium cations. The determination of N-methyl-N-propylpiperidinium (PMPI) and N-butyl-N-methylpiperidinium (BMPI) was achieved by reversed-phase ion-pair chromatography with indirect ultraviolet detection using 0.5 mmol/L 1-ethyl-3-methylimidazolium tetrafluoroborate–0.1 mmol/L sodium 1-heptanesulfonate aqueous solution/methanol (volume ratio 70–30) as mobile phase. The retention times of PMPI and BMPI were 1.96 and 2.48 min, respectively. The limits of detection for PMPI and BMPI were 0.29 and 0.53 mg/L, respectively. The linear range of the method for determination of PMPI and BMPI was 2.0–100.0 mg/L. The relative standard deviation of the method is less than 2.0%. The method has been applied to the determination of piperidinium ionic liquid samples synthesized by chemistry laboratory with satisfactory accuracy and precision. This method is simple, rapid and easy to popularize.

The effect of column packing procedure on column end efficiency and on bed heterogeneity – Experiments with flow-reversal

The effect of column end structure and bed heterogeneity of six commercially available reversed-phase chromatographic columns for fast liquid chromatography with different column packing materials – such as fully porous (Waters XBridge C18 with 1.7 μm particles) and superficially porous (Waters CORTECS C18 with 1.6 μm particles), with column dimension of 2.1 × 50, 100 or 150 mm were tested with flow-reversal method. The method includes arresting the flow when a non-retained marker (thiourea) has penetrated to a given distance into the column and then reversing the column. Hence, when the flow has been restarted, the sample is eluted at the same end of the column where it entered. The experiments showed that all columns are axially heterogeneous, and some differences could be observed between the two respective column ends. Furthermore, we can conclude that the shorter columns are axially more homogeneous than the longer ones, thus the column length is an influencing factor on the column packing procedure.

Investigation into reversed phase chromatography peptide separation systems part II: An evaluation of the robustness of a protocol for column characterisation

The robustness of the Peptide Reversed Phase Chromatography (RPC) Column Characterisation Protocol was evaluated using reduced factorial design, to ascertain the degree of control required for parameters including temperature, flow rate, dwell volume, a systematic shift in the gradient, amount of formic acid in the aqueous and organic, pH of the ammonium formate and amount of acetonitrile (%MeCN) in the strong solvent, where a loss of MeCN resulted in an unacceptable variation. Mitigations have been introduced to ensure the integrity of the data to allow RPC columns to be characterised using peptides as probes, with the definitive protocol described. In addition, the instrument and column batch to batch variability were assessed with good reproducibility.

A novel two-dimensional liquid chromatography system for the simultaneous determination of three monoterpene indole alkaloids in biological matrices

The present paper describes a novel two-dimensional liquid chromatography (2D-LC) system, which is comprised of a first-dimensional ion exchange chromatography (IEX1) column, trap column, and second-dimensional reversed-phase chromatography (RP2) column system. The biological sample is separated by the first-dimensional LC using an IEX column to remove interferences. The analytes are transferred to the trap column after heart-cutting. Then, the analytes are transferred to the second-dimensional LC using an RP2 column for further separation and ultraviolet detection. This 2D-LC system can offer a large injection volume to provide sufficient sensitivity and exhibits a strong capacity for removing interferences. Here, the determination of three monoterpene indole alkaloids (MIAs; gelsemine, koumine, and humantenmine) from Gelsemium in biological matrices (plasma, tissue, and urine) was used this 2D-LC system. After a rapid and easy sample preparation method based on protein precipitation, the sample was injected into the 2D-LC. The method was developed and validated in terms of the selectivity, LOD, LOQ, linearity, precision, accuracy, and stability. The sample preparation time for the three MIAs was 15min. The LOD for these compounds was 10ng/mL, which was lower than the developed HPLC methods. The results showed that this method had good quantitation performance and allowed the determination of gelsemine, koumine, and humantenmine in biological matrices. The method is rapid, exhibits high selectivity, has good sensitivity, and is low-cost, thus making it well-suited for application in the pharmaceutical and toxicological analysis of Gelsemium. Graphical abstract.

Recovery of gallium from wafer fabrication industry wastewaters by Desferrioxamine B and E using reversed-phase chromatography approach

Gallium (Ga) is a critical element in developing renewable energy generation and energy efficient systems. The supply of Ga is at risk and needed recycling technologies for its availability in future. This study demonstrated the recovery of Ga3+ from low gallium concentrated wafer fabrication industry wastewaters using the siderophores desferrioxamine B (DFOB) and desferrioxamine E (DFOE). The complexation of Ga3+ by DFOB and DFOE was through hydroxamate group as demonstrated by infrared spectroscopy, nuclear magnetic resonance and density functional theory calculations. The high selectivity of DFOB/E towards Ga3+ was observed due to the formation of highly stable complex. Indeed, due to the formation of such high stability complex, the DFOB and DFOE were able to successfully complex 100% Ga in the two different process water from wafer fabrication industry. For the recovery of the siderophores, a high rate of decomplexation of Ga (>90%) was achieved upon addition of 6 times excess of ethylenediaminetetraacetic acid (EDTA) at pH of 3.5. More than 95% of Ga-DFOB and Ga-DFOE complex were recovered with purity (% of Ga moles in comparison to total moles of metals) of 69.8 and 92.9%, respectively by application of a C18 reversed-phase chromatography column. This study, for the first time, demonstrated a technical solution to the recovery of Ga3+ from the low concentrated wastewater based on siderophores and reversed-phase chromatography. A German patent application had been filed for this technology.

Design of Reversed-Phase Chromatography Column Packing Material for Protein Separation: Part 2- Chromatographic Performance

In the second part of the two-part study, monodisperse and porous poly(vinyl acetate-co-divinylbenzene) [poly(VAc-co-DVB)] and poly(divinylbenzene) [poly(DVB)] column packing materials synthesized in the first part of the study were tested in Reverse Phase Chromatography (RPC) under different conditions. The investigation of column performances concluded that columns packed with poly(VAc-co-DVB) (2500 plate/m) and poly(DVB) (1500 plate/m), showed high and stable theoretical plate number and high peak resolution values compared to literature values. Protein recovery data in RPC column, poly(VAc-co-DVB) (93-99%), were determined as similar as quantitative values for all proteins. Reproducibility tests of analytes of poly(VAc-co-DVB) column were assessed "run to run" and "day to day" Relative Standard Deviation (RSD) values were determined under 2.8%.

Homo- and hetero-difunctionalized β-cyclodextrins: Short direct synthesis in gram scale and analysis of regiochemistry.

The regioselective difunctionalization of cyclodextrins (CDs) leading to derivatives amenable to further transformations is a daunting task due to challenging purification and unambiguous characterization of the obtained regioisomers with similar physicochemical properties. The primary-side homo-difunctionalization of β-CD can lead to three regioisomers, while the hetero-difunctionalization can generate three pairs of pseudoenantiomers. Previously, approaches with several synthetic steps, expensive reagents, high purification demands and low yields of the products have been employed. Herein we present direct, short and efficient primary-side difunctionalization strategies featuring reproducibility, ease of product purification, scalability of the reactions and versatility of the substituents introduced. Specifically, the prepared ditosylated β-CDs were separated using preparative reversed-phase column chromatography and their structures were elucidated by NMR experiments. Azidation led to the corresponding pure diazido regioisomers. Direct monotosylation of 6-monoazido-β-CD or monoazidation of the single regioisomers 6A,6X-ditosyl-β-CDs afforded hetero-difunctionalized 6A-monoazido-6X-tosyl-β-CDs in significant yields. Overall, the single regioisomers, 6A,6X-ditosyl-, 6A,6X-diazido- and 6A-monoazido-6X-monotosyl-β-CD were prepared in one or two steps and purified in multigram scale thus opening the way towards further selective and orthogonal functionalizations of β-CD hosts.

Optimization of column chromatography technique for the isolation of tetrandrine from Cyclea peltata and LC-ESI-MS based quantification and validation of the method

Reverse phase column chromatography technique was employed to isolate tetrandrine from methanolic extract of Cyclea peltata roots. Tetrandrine was efficiently isolated using 0.02% aqueous diethylamine and methanol (25:75, v/v) as mobile phase with a purity of 98.63%. Compound characterisation was achieved using high-performance liquid chromatography coupled with electrospray ionization mass spectrometry and other spectroscopic methods. In addition, quantification of tetrandrine and validation of the method was carried out following International Conference on Harmonisation guidelines. The study provides a simple and cost effective method to isolate substantially good quantity and pure tetrandrine from Cyclea peltata.

Development of a screening and confirmatory method for the analysis of polar endogenous compounds in saliva based on a liquid chromatographic-tandem mass spectrometric system

In this paper, a high-throughput approach is proposed for the sensitive screening and the confirmatory analysis of polar compounds in saliva using a two-step approach based on a liquid chromatographic system coupled to a triple quadrupole mass spectrometer. A reversed-phase chromatographic column was used in both steps and changes in the composition of the mobile phase allowed the screening and the confirmatory analyses to be performed with the same instrumental configuration. The proposed strategy has been tested for the determination of a multiclass group of polar endogenous compounds (creatinine, polyamines and amino acids) in saliva samples. The validation of the entire procedure showed consistent results for all the compounds in both steps. Repeatability and reproducibility were evaluated for both procedures, with values below 8% in the case of repeatability and 17% in the case of reproducibility. The instrumental limits of detection were found to be between 1.22 × 10−3 and 46.1 × 10−3 mg/L for creatinine and lysine, respectively, and accuracy of the method was evaluated in terms of apparent recoveries and values were found to be between 80 and 127%. Matrix effects were evaluated and it was found that the analytical outcome was influenced by the matrix of the sample. Thus, a one-point standard addition method was used for quantification. The optimized two-step procedure was applied to saliva samples from apparent healthy volunteers. Overall, satisfactory results were obtained in both steps, demonstrating its applicability for quantitative analysis of polar endogenous compounds in this kind of matrices.

Studies on the chemical compositions of Hyptis suaveolens (L.) Poit

This study aimed at identifying the chemical compounds isolated from the aerial parts and roots of Hyptis suaveolens (L.) Poit. The compounds were isolated and purified by a combination of various chromatographic techniques including silica gel column chromatography, silica gel reverse phase column chromatography, Sephadex LH-20 gel column chromatography, semi-preparative HPLC and recrystallization. The chemical structures were analyzed and elucidated based on physiochemical properties and spectroscopic analysis. From the aerial parts of H. suaveolens, eight compounds were isolated and identified as quercetin 3-O-?-D-glucopyranoside (1), apigenin (2), sorbifolin (3), quercetin (4), kaempferol (5), genkwanin (6), rosmarinic acid (7) and methyl rosmarinate (8). Two compounds were isolated from the roots of H. suaveolens and identified as podophyllotoxin (9) and picropodophyllotoxin (10). Compounds 2?6 were isolated from H. suaveolens for the first time while compound 10 was isolated from the genus of Hyptis for the first time. The results of this study provided a scientific basis for the development of the medicinal value of H. suaveolens and have important theoretical significance for the chemical utilization of H. suaveolens resources.

A new triterpene diglycoside from the roots of Bupleurum chinense DC. and its inhibitory effect on adipogensis in 3T3-L1 cells

A new triterpene diglycoside, tibesaikosaponin V (TKV), was isolated from the methanol extract of the roots of Bupleurum chinense DC. by normal and reversed phase column chromatography. The chemical structure of this new compound was elucidated based on extensive spectroscopic analysis, including 1D and 2D NMR, HR-ESI-MS, and chemical degradation method. Our results indicated that TKV inhibited lipid accumulation and triacylglycerol content occurred without cytotoxicity to 3T3-L1 adipocytes. Furthermore, TKV significantly suppressed the mRNA expression of nuclear transcription factors such as, peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer binding protein α (C/EBPα). These results suggest that TKV shows the ability to inhibit 3T3-L1 preadipocyte differentiation of and may have therapeutic potential for obesity and its associated metabolic disorders.

An application of QSRR approach and multiple linear regression method for lipophilicity assessment of flavonoids

The analysis of quantitative structure-retention relationships (QSRR) is useful tool for assessment of compound’s lipophilicity/hydrophobicity due to similarity between its retention in chromatographic system and ability to permeation through biological membranes. The main goal of this study was to compare usefulness of two reversed-phase chromatographic columns (Synergy POLAR and Synergy-FUSION) for lipophilicity assessment of 30 structurally diverse flavonoids using the QSRR approach and multiple linear regression method. The developed MLR models included the mechanistically interpretable geometrical descriptors: 3D Molecule Representation of Structure based on Electron diffraction (3D-MoRSE) and Radial Distribution Function (RDF). Both models were evaluated by the internal and external validation and selected descriptors were further interpreted. According to obtained results the FUSION-RP column can be recommended to log kw prediction of flavonoids. The comprehensive interpretation of molecular descriptors was used to present the molecular mechanisms and structural features governing the chromatographic retention of tested compounds.

Therapeutic Drug Monitoring of Lacosamide by LC-MS/MS.

High-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) has become a primary analytical methodology in therapeutic drug monitoring of antiepileptic drugs (AEDs). To demonstrate the utility of LC-MS/MS in measuring drug concentrations in serum or plasma, analysis of lacosamide (Vimpat™) is discussed in this chapter. Lacosamide is an example of the newer-generation AEDs. The drug is extracted by protein precipitation and dilution of the serum specimen. A small volume of the extracted specimen is injected into a reversed-phase chromatography column, and lacosamide is identified by positive electrospray ionization (ESI) mass spectrometry in the multiple reaction monitoring (MRM) mode, which provides selectivity for quantitative analysis. A deuterated internal standard is used to correct for any loss of analyte during the process of extraction and analysis. A seven-point calibration curve and two levels of quality controls are included in each batch.

New insights into phenol and polyphenol composition of Stevia rebaudiana leaves

The diversity in phenols and polyphenols of stevia leaf has been simplified applying sequential fractionation techniques on its ethanol extract through ultrasound assisted maceration. Two of the fractions obtained by reverse-phase column chromatography resulted differently active in an extensive antioxidant and cytotoxic screening. Both fractions were chemically profiled by ultra-performance liquid chromatography (UHPLC) coupled with electrospray ionization (ESI) quadrupole/time-of-flight (QqTOF) mass spectrometry (MS). One of the fractions was composed mainly of chlorogenic acids and flavonol triglycosides, whereas the other was rich in flavonoids mono- and diglycosides and in their hydroxycinnamoyl derivatives. Among the fifty compounds identified, non-phenol metabolites, such as benzyl primeveroside and roseoside, as well as a lignan polyphenol (5′), are reported for the first time as constituents of the Stevia leaf.