High-performance Liquid Chromatography Column Research Articles

A new aquatic pathogen inhibitor produced by the marine fungus Aspergillus sp. LS116

In this study, a fungal strain LS116 with antibacterial activity against aquatic pathogen Vibrio harveyi was screened out from sponge-associated fungi by Oxford cup method. According to its morphological characteristics and internal transcribed spacer (ITS) analysis, the active strain belonged to the genus Aspergillus. A bioactivity-guided approach was conducted to identify the antibacterial substances in the culture extract of strain LS116, a new C23 steroid with bicyclo[4.4.1]A/B ring, named aspergillsteroid A (1), and a known C25 steroid with the same structure skeleton, neocyclocitrinol B (2), were isolated and purified by column chromatographs and HPLC (high-performance liquid chromatography) for the first time. Their chemical structures were determined by HRESIMS (high-resolution electrospray ionization mass spectroscopy) spectrometry, NMR (nuclear magnetic resonance) spectroscopy, and CD (circular dichroism). Among them, compound 1 displayed significant antibacterial activity against V. harveyi with a MIC value of 16 μg/mL. The study demonstrated that compound 1 can be considered as a new promising agent for the control of aquatic disease in the future.

Mixed polymer brush as a functional ligand of silica beads for temperature-modulated hydrophobic and electrostatic interactions

We developed a mixed polymer brush, which consists of temperature-responsive polymer and cationic polymer modified beads as functional chromatographic matrices, for temperature-modulated multiple hydrophobic and electrostatic interactions. The mixed polymer brush was modified on silica beads through the combination of reversible addition−fragmentation chain transfer (RAFT) polymerization of N,N-dimethylaminopropyl acrylamide (DMAPAAm) and surface initiated atom transfer radical polymerization (ATRP) of N-isopropylacrylamide (NIPAAm). Zeta potential measurement of the mixed polymer brush revealed that the zeta potential increased with increasing temperature, which was attributed to the exposed PDMAPAAm on the beads arising from the shrinking of PNIPAAm upon increasing the temperature. The prepared beads were used as the packing material of high performance liquid chromatography (HPLC) columns, and the elution behavior of steroids, adenosine nucleotides, and proteins through the column was observed. The retention time of steroids increased with increasing the column temperature because of the dehydration of PNIPAAm in the mixed polymer brush. Adenosine nucleotides were also retained by the columns, which was attributed to the electrostatic interaction with PDMAPAAm in the mixed polymer brush. Several proteins were adsorbed on the column at elevated temperatures because of the enhanced electrostatic interaction of exposed PDMAPAAm and the enhanced hydrophobic interaction resulting from the dehydration of PNIPAAm. By exploiting this unique property, mixtures of proteins could be separated by simply changing the column temperature. These results indicate that the developed mixed polymer brush modified beads would be useful as functional chromatographic packing matrices for thermally-modulated multiple hydrophobic and electrostatic interactions.

What is the influence of two strain rates on the relationship between human cortical bone toughness and micro-structure?

Cortical bone fracture mechanisms are well studied under quasi-static loading. The influence of strain rate on crack propagation mechanisms needs to be better understood, however. We have previously shown that several aspects of the bone micro-structure are involved in crack propagation, such as the complete porosity network, including the Haversian system and the lacunar network, as well as biochemical aspects, such as the maturity of collagen cross-links. The aim of this study is to investigate the influence of strain rate on the toughness of human cortical bone with respect to its microstructure and organic non-collagenous composition. Two strain rates will be considered: quasi-static loading (10-4 s-1), a standard condition, and a higher loading rate (10-1 s-1), representative of a fall. Cortical bone samples were extracted from eight female donors (age 50-91 years). Three-point bending tests were performed until failure. Synchrotron radiation micro-computed tomography imaging was performed to assess bone microstructure including the Haversian system and the lacunar system. Collagen enzymatic cross-link maturation was measured using a high performance liquid chromatography column. Results showed that that under quasi-static loading, the elastic contribution of the fracture process is correlated to both the collagen cross-links maturation and the microstructure, while the plastic contribution is correlated only to the porosity network. Under fall-like loading, bone organization appears to be less linked to crack propagation.

Validation of rapid RP-HPLC method for concurrent quantification of amlodipine and celecoxib in pure and formulation using an experimental design

Objective of the proposal was to establish rapid, economical, and robust liquid chromatographic procedure to perform quality control of a pharmaceutical preparation containing amlodipine and celecoxib using an experimental design. The proposed new high-performance liquid chromatography (HPLC) procedure involves isocratic separation using sodium phosphate buffer (pH 5.6): acetonitrile: methanol in a ratio 30:55:15 (v/v) as the mobile phase on a Zorbax C18 (150 mm) reverse-phase (RP)-HPLC column. The analysis was performed at 25 °C, using a flow rate of 1.2 mL/minute. The column elutes were detected at 239 nm and etoricoxib has been selected as an internal standard (IS). The calibration curve exhibited good linearity in the range of 5–30 µg/mL of amlodipine and 50–500 µg/mL of celecoxib with a good correlation coefficient (r2 > 0.999). The precision and accuracy were presented in terms of%RSD and%RE and were in an acceptable range (<2). The accuracy of the suggested procedure was established by the results of recovery studies, executed using a standard addition method. A robustness test was performed using fractional factorial design with three major contributing factors. The pH of the mobile phase and flow rate demonstrated a substantial effect on the peak area ratio of amlodipine, whereas the percent of acetonitrile and the flow rate together exhibited an effect on the peak area ratio of celecoxib. This validated, robust analytical method has been utilized to quantify amlodipine and celecoxib from formulations with large concentration differences. Further, the comparison of results with reported methods indicated that no significant difference was observed in terms and accuracy and precision.

Versatile MOF separates drug molecule enantiomers

In drug discovery, separating enantiomers of a compound usually involves high-performance liquid chromatography (HPLC) columns packed with specialized chiral stationary phases. These columns are usually compatible with only polar or nonpolar solvent mixtures, meaning chemists sometimes have to switch between columns to find the right solvent system. Now, a team of researchers has demonstrated a reusable and scalable metal-organic framework (MOF) that can do chiral separations with both polar and nonpolar solvents (J. Am. Chem. Soc. 2019, DOI: 10.1021/jacs.9b06500). Jose Ramon Galan-Mascaros of the Institute of Chemical Research of Catalonia and colleagues synthesized a water-stable MOF composed of copper atoms linked with modified l-histidine molecules. Galan-Mascaros’s lab teamed up with Melissa M. Reynolds of Colorado State University and other collaborators and showed that an HPLC column packed with this MOF could separate the enantiomers of the pain-relieving drug ibuprofen and the infamous medication...

Retention behavior of ginsenosides in a sulfo-based high performance liquid chromatography column

We herein report the use of a sulfo-based column and hydrophilic interaction chromatography (HILIC) to separate 14 ginsenosides, namely Rb1, Rb2, Rb3, Rc, Rd, Rf, Re, Rg1, Rg2, Rg3, Rh1, Rh2, F2, and C-K. In addition to its rapid and efficient ability to separate these ginsenosides, the sulfo-based column exhibited a good relationship between the ginsenoside capacity factor (k′) and molecular weight (Mw) and a strict elution order corresponding to the polarity (P) of the ginsenosides, as confirmed by thin layer chromatography.

Endocrine disrupting Bisphenol A detection in different water samples in Iraq

Poly-carbonate plastics containing endocrine disruptor BPA (Bisphenol A), it have been used in the production process of water bottles, food products containers to storage, infants feeding milk bottles and raw material for some medical supplies. Trace quantity of bisphenol has been measured in water-samples that kept in poly-carbonate containers. It has been recognized that bisphenol a can be generated from poly-carbonate containers and then transfer into stored food. Such transfer is accelerated by leaving the containers in sunlight for long time. In this study a simple and rapid method was established to quantify BPA using high performance liquid chromatography (HPLC) in water samples in Al-Diwaniah city, Iraq. The chromatographic HPLC separation includes, extraction then detection of BPA using C18 as a HPLC column, followed by quantitative determination BPA using pre-measured known concentration as a standard. The analysis method shows a recovery value around 85% ± 2.9. A suitable sensitivity value was obtained with an instrumental limit of detection and quantification (LOD and LOQ) ranged from 33 to 72 µgL−1. The detection of BPA was effectively achieved using homemade and commercially supplied samples. The obtained numbers were inside the allowable concentration

Salting-out assisted liquid-liquid extraction coupled with high-performance liquid chromatography for the determination of vitamin D3 in milk samples.

In this study, salting-out assisted liquid–liquid extraction (SALLE) as a simple and efficient extraction technique followed by high-performance liquid chromatography (HPLC) was employed for the determination of vitamin D3 in milk samples. The sample treatment is based on the use of water-miscible acetonitrile as the extractant and acetonitrile phase separation under high-salt conditions. Under the optimum conditions, acetonitrile and ammonium sulfate were used as the extraction solvent and salting-out agent, respectively. The vitamin D3 extract was separated using Hypersil ODS (250x i.d 4.6 mm, 5 µm) HPLC column that was coupled with diode array detector. Vitamin D2 was used as internal standard (IS) to offset any variations in chromatographic conditions. The vitamin D3 and the IS were eluted in 18 min. Good linearity (r2 > 0.99) was obtained within the range of 25–600 ng g−1 with the limit of detection of 15 ng g−1 and limit of quantification of 25 ng g−1. The validated method was applied for the determination of vitamin D3 in milk samples. The recoveries for spiked samples were from 94.4 to 113.5%.

A new demethyl abietane diterpenoid from the roots of Tripterygium wilfordii

A new demethyl abietane diterpenoid, Triptotin K (3) together with three known compounds, friedelin (1), canophyllal (2), and triptonoterpene (4) were isolated from the roots of Tripterygium wilfordii Hook. f. by silica gel column and preparative high performance liquid chromatography. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Triptotin K showed cytotoxic activities against KB, KBv200, HepG2, and MCF-7/ADM cells lines with IC50 values of 29.88, 36.50, 39.55, and 41.38 μM, respectively.

6-(2-Formyl-5-methyl-1H-pyrrol-1-yl)hexanoic Acid as a Novel Retronasal Burnt Aroma Compound in Soy Sauce Aroma-Type Chinese Baijiu.

Two-step high-performance liquid chromatography (HPLC) separation in combination with taste dilution analysis was used to screen the retronasal burnt aroma compounds in soy sauce aroma-type Chinese Baijiu (Chinese traditional liquor). A major retronasal burnt aroma region was purified by Sephadex LH-20 column chromatography and HPLC separation from the soy sauce aroma-type Baijiu sample. A novel retronasal burnt aroma compound was identified as 6-(2-formyl-5-methyl-1H-pyrrol-1-yl)hexanoic acid (1) by means of high-resolution mass spectrometry and 1D/2D nuclear magnetic resonance. The retronasal burnt aroma threshold of compound 1 was determined to be 209.5 μg/L in 53% ethanol aqueous solution. Quantitative analysis showed that the concentration of compound 1 ranged from below the limit of quantitation (3.8 μg/L) to 224.2 μg/L in the Baijiu samples. Moreover, the sensory evaluation also revealed that compound 1 may contribute to the retronasal burnt aroma of the Baijiu at its subthreshold concentration by the additive effect with the recently reported retronasal burnt aroma compound 2-hydroxymethyl-3,6-diethyl-5-methylpyrazine.

Development and validation of a rapid HPLC method for multiresidue determination of erythromycin, clarithromycin, and azithromycin in aquaculture fish muscles

An accurate, sensitive, and reproducible high-performance liquid chromatographic method with diode array detection has been developed for simultaneous determination of erythromycin, clarithromycin, and azithromycin residues in fish muscles. Analysis was carried out using a Shodex Asahipak high-performance liquid chromatography (HPLC) column, monitoring was at 210 nm and a mobile phase consisting of a mixture of acetonitrile and phosphate buffer (pH 11 ± 0.05) in the ratio of 60:40 (v/v). Solid-phase extraction method was used in samples extraction and purification. Recoveries were in the range 72.0–92.2% with relative standard deviation (RSD) from 2.3% to 8.3%. This method was validated for fish muscles in aquaculture following the commission decision 2002/657/EC criteria. It is demonstrated that the new method is robust for detection and quantification of the three macrolides residues. Decision limit (CCα) was from 214 to 228 μg/kg and capacity of detection (CCβ) was from 228 to 256 μg/kg.

The effect of temperature on the separation of enantiomers with coated and covalently immobilized polysaccharide-based chiral stationary phases

This article describes our attempt to re-visit the role of temperature in the separation of enantiomers with polysaccharide-based chiral columns in high-performance liquid chromatography (HPLC). Rarely observed increased retention and separation factors with increasing temperature, as well as temperature dependent reversal of enantiomer elution order are reported for several arylpropionic acid derivatives. Chiral columns with coated and covalently immobilized chiral selectors were compared from the viewpoint of effect of temperature on analyte retention, enantiomer separation and enantiomer elution order. Thermodynamic parameters were calculated for analyte transfer from the liquid phase to the chiral stationary phase and the effect of temperature on chiral selectors was investigated by using differential scanning calorimetry (DSC). DSC results along with chromatographic studies indicate that polysaccharide-based chiral selectors undergo some kind of transition at elevated temperature that is not reversible in the thermodynamic sense of this term.

Preparation of Poly-(GMA-EDA-β-CD-co-TMPTMA) Monolith as High Performance Liquid Chromatography Chiral Stationary Phase Column

An enantiomer molecule consisted of the chiral atom has different structure conformations, which exhibit different activities as well. Yet, its separation considerably difficult since ordinary separation could not separate both molecules. One of the popular enantioseparations which are often used was using organic polymer monolithic column modified by ethylenediamine-β-cyclodextrin (EDA-β-CD) as the enantioseparations site. The aim of this research was to produce chiral stationary phase column for enantioseparations of (±)-citronellal. It was conducted by preparing monolithic column using monomer glycidyl methacrylate (GMA), trimethylolpropane trimethacrylate (TMPTMA) as crosslinker, 1-propanol, 1,4-butanediol, and water as pore-forming agents (porogens) in the presence of α,α'-azoisobutyronitrile (AIBN) as radical initiator inside polyetheretherketone (PEEK) tubing. It was then modified with EDA-β-CD synthesized from β-CD. Finally, it was installed as a high-performance liquid chromatography column. The result shows the produced chiral stationary phase column could separate (±)-citronellal at a retention time of 44.76 and 45.71 min.

14-Deoxy-11,12-Didehydroandrographolide: A novel compound isolated from Andrographis paniculata Nees. induces robust apoptosis in leukemic cells

Objective: Andrographis paniculata is widely cultivated in South and Southeast Asian countries and popularly used in “Ayurveda” medicine. We attempted to investigate antileukemic activity of the biomolecules extracted from this plant and a probable mechanism of action. Materials and Methods: Biomolecules from methanolic extract were isolated using silica gel column chromatography and high-performance liquid chromatography. The structures were determined by liquid chromatography-mass spectrometry (LC-MS), 1H nuclear magnetic resonance (NMR), and 13C NMR. In vitro antiproliferative activity was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The apoptotic efficacy of the most potent molecule was investigated by annexin V and propidium iodide (V/PI) staining and flow cytometry. Caspase activation, cell cycle distribution pattern, and nuclear morphology of the treated leukemic cells were also investigated. Results: From the methanolic extract, four biomolecules were isolated, namely 14-deoxy-11,12-didehydroandrographolide (1), andrographolide (2), neoandrographolide (3), and 14-deoxy-11,12-didehydroandrographiside (4). Results of MTT assay exhibited that out of four compounds, compound (1) showed the most potent activity against all the cell lines tested with the lowest IC50 values of 13 μM on U937 cells. Annexin V/PI staining revealed that the compound was able to induce apoptosis in concentration-dependent manner with IC50 value being 17.66 μM. Apoptotic induction was mediated through elevated activation of caspase-3 and caspase-9. Cell cycle analysis revealed that the compound (1) effectively increased the sub-G0-G1 population in the treated U937 cells (73.25% at 50 μM) in comparison to control set (3.12%). DAPI nuclear staining indicated that compound (1) increased the number of deformed nuclei and an increased level of apoptotic body formation in the treated cells.

Simultaneous Determination of Moxifloxacin and Flavoxate by RP-HPLC and Ecofriendly Derivative Spectrophotometry Methods in Formulations.

Simple, fast, and precise reversed-phase (RP)-high-performance liquid chromatography (HPLC) and two ecofriendly spectrophotometric methods were established and validated for the simultaneous determination of moxifloxacin HCl (MOX) and flavoxate HCl (FLX) in formulations. Chromatographic methods involve the separation of two analytes using an Agilent Zorbax SB C18 HPLC column (150 mm × 4.6 mm; 5 µm) and a mobile phase consisting of phosphate buffer (50 mM; pH 5): methanol: acetonitrile in a proportion of 50:20:30 v/v, respectively. Valsartan was used as an internal standard. Analytes were monitored by measuring the absorbance of elute at 299 nm for MOX and 250 nm for FLX and valsartan. Two environmentally friendly spectrophotometric (first derivative and ratio first derivative) methods were also developed using water as a solvent. For the derivative spectrophotometric determination of MOX and FLX, a zero-crossing technique was adopted. The wavelengths selected for MOX and FLX were −304.0 nm and −331.8 nm for the first derivative spectrophotometric method and 358.4 nm and −334.1 nm for the ratio first-derivative spectrophotometric method, respectively. All methods were successfully validated, as per the International Conference on Harmonization(ICH) guidelines, and all parameters were well within acceptable ranges. The proposed analytical methods were successfully utilized for the simultaneous estimation of MOX and FLX in formulations.

Determination of progesterone compounds in the crude methanol extract of benalu duku leaves.

Background and Aim:Dendrophthoe pentandra L. Miq (benalu duku) is a parasitic herb that commonly grows on the host plant Lansium domesticum. Researchers have found that the plant contains anticancer compounds and may contain phytoandrogens, including progesterone-like compounds, in its crude methanol extract. The objective of the current study was to investigate the compound of phyto progesterone in benalu duku leaves after extracted by methanol and prepared using an analytical column of high-performance liquid chromatography (HPLC).Materials and Methods:About 400 g of benalu duku leaves were pulverized, and their compounds were isolated by the isocratic method using an RP-18 analytical column (5 µm) with a mobile phase of 70:30 (methanol: water) in a photodiode array detector adjusted to 254 nm. The phyto progesterone compound was identified at a retention time of approximately 6.01 min.Results:By LC-electrospray ionization mass spectrometry focusing on molecular fractions, the fingerprint area of the Fourier transform-infrared spectroscopy (FT-IR, cm−1) and Hnuclear magnetic resonance (NMR) spectra indicated that the phyto progesterone product isolated was identical to the certified reference material of pure progesterone, particularly the specific functional groups in the FT-IR spectrum at wavenumbers of 1317.43 cm−1 and 1386.86 cm−1 and in the proton HNMR spectrum at carbon 21 of progesterone (p<0.05).Conclusion:Each 49.888 µg/mL of crude benalu duku leaf extract dissolved in the mobile phase contained 28.515±0.713 µg/mL phyto progesterone.

Determination of Genotoxic melamine impurity in Allopurinol by High performance liquid chromatography

A rapid, sensitive and highly selective High performance liquid chromatography (HPLC) method was developed and validated for the determination of trace level Genotoxic Melamine impurity, as required by USFDA and that may be present in Allopurinol. HPLC column used was Cosmosil C18 (250 x 4.6 mm, 5µm). The isocratic elution modewas selected where the mobile phaseconsists of 1.25 g/l Sodium dihydrogen phosphate solution. The flow rate was set to1.0 mL/minand run time of 30 minutes. UV detector with wavelength 208 nm was used for theanalysis. The column oven temperature was kept at 25°C. The developed method was validated according to ICH guideline and found to be linear in the range of 5.20 ppm to 52.05 ppm with a correlation coefficient 0.9997. Limit of detection and limit of quantitation was found to be 1.21 ppm and 3.68 ppm respectively. Recovery for this impurity was found between 93.37% and 107.59%.A method was found to be specific, selective, precise, and robust. The developed method can successfully be applied for the determination of Genotoxic Melamine impurity in Allopurinol.

Preparation of a poly(1, 6-hexylene dimethacrylate) conventional size high performance liquid chromatographic monolithic column for separation of small molecules

A porous monolithic column was prepared by in situ free-radical polymerization in a stainless steel column (4.6 mm i.d. × 100 mm). The polymerization reacted using 1,6-hexylene dimethacrylate as single crosslinking monomer, methanol and 1-dodecanol as the binary porogens and 2,2-azobisisobutyronitrile asinitiator. During the column preparation process, the ratio of monomer to porogen was optimized to obtain the best column. The chemical groups and morphology of monolithic column were characterized by the Fourier transform infrared spectroscopy and scanning electron microscopy, respectively. Furthermore, the influence of mobile phase composition and flow rate on the separation of aromatic compounds was investigated.The results showed that the poly(1,6-hexylene dimethacrylate) monolithic column had high mechanical stability at flow rate of 4 mL/min. Phenol, 1-naphthol, biphenyl and phenanthrene were easily separated on the prepared monolithic column using acetonitrile/water (58/42, v/v) as mobile phase at flow rate of 2 mL/min. The relative standard deviation of run-to-run and column-to-column of the retention times for the four standard compounds were <0.3% and 3.3%, respectively. Three kinds of small molecules (phenols, aromatic hydrocarbons, aromatic acids) were successfully separated on the poly(1,6-hexylene dimethacrylate) monolithic column. The possible chromatogramphic retention mechanism on poly(1,6-hexylene dimethacrylate) monolithic column was determined as hydrophobic interaction and dipole-dipole interaction. The conventional size poly(1,6-hexylene dimethacrylate) monolithic column has potential to separate small molecule compounds rapidly by high performance liquid chromatography.

The different dissipation behavior of chiral pesticide paclobutrazol in the brine during Chinese cabbage pickling process.

Paclobutrazol (PBZ) is a kind of chiral pesticide, which is a plant growth regulator and has fungicidal activity. Because of the steric-hindrance effect, there are two enantiomers (2S, 3S; 2R, 3R) in the production. This research studied on the dissipation behavior of chiral pesticide PBZ in the brine during the Chinese cabbage pickled process by phase column-high performance liquid chromatography (HPLC). The result demonstrated the PBZ enantiomers had the different dissipation in the brine. The study on the behavior of chiral pesticide PBZ in food may provide more sufficient data and information for understanding the potential risk in food and evaluating the environmental pollution at the enantiomer level.

A systematic study of determination and validation of finasteride impurities using liquid chromatography

Pharmaceutical use of finasteride (Dilaprost®) has been well documents in the peer-reviewed literature; however, the presence of trace amounts of related substances (impurities) in finasteride may influence the tharapeutic efficacy and safely. Due to limited information available, the objective of this study was to develop a quantification method for the three impurities of finasteride using high performance liquid chromatography (HPLC) with an ultraviolet (UV) detector. The compounds (impurities) of finasteride that are registered with the European Pharmacopeia, which we sought to validate are: -N-(1,1-dimethylethyl)-3-oxo-4-aza-5α-androstane-17β-carboxamide (impurity A), methyl 3-oxo-4-aza-5α-androst-1-ene-17β-carboxylate (impurity B), and -N-(1,1-dimehylethyl)-3-oxo-4-azaandrosta-1,5-diene-17β-carboxamide (impurity C). Analyses were performed using a Nova Pac C18 column for HPLC with isocratic elution. Detection was carried out at 210 nm, the concentration of the three impurities was in the range was 1.5–4.5 μg mL−1 at ambient temperature with a mobile phase of water + acetonitrile + tetrahydrofuran (80:10:10, v/v/v) and the flow rate was 2.0 mL min−1. The recoveries were: 101.35 ± 0.62% (impurity A), 101.60 ± 2.66% (impurity B) and 101.97 ± 2.05% (impurity C). Validation of the method yielded fairly good results as it relates to the precision and accuracy. It is, therefore, concluded that the method would be suitable for not only the separation and determination of processed impurities to monitor the reactions, but also for the quality assurance of finasteride and its related substances.