Application Of HPLC Research Articles

Application of biomimetic HPLC to estimate lipophilicity, protein and phospholipid binding of potential peptide therapeutics

Peptide therapeutics are new modalities offering several challenges to drug discovery. They are generally less stable and permeable in vivo. The characterization of their lipophilicity cannot be carried out using the traditional in silico or wet octanol/water partition coefficients. The prediction of their in vivo distribution and permeability is also challenging. In this paper, it is demonstrated that the biomimetic properties such as lipophilicity, protein and phospholipid binding can be easily assessed by HPLC using chemically bonded protein and immobilized artificial membrane (IAM) stationary phases. The obtained properties for a set of potential therapeutic peptides with 3 to 33 amino acids have been analysed and it was found that similar characteristics of the properties could be observed as for small molecule drugs. The albumin binding showed correlation with their measured lipophilicity on the C-18 stationary phase with acidic peptides showing stronger than expected albumin binding. The (IAM) chromatography revealed peptide membrane affinity, which was stronger for positively charged peptides (containing arginine) and showed correlation to the alpha-1-acid glycoprotein (AGP) binding, which was also stronger for positively charged compounds. The in vivo volume of distribution and drug efficiency of the peptides have been estimated using the models developed for small molecules. One of the candidate linear peptides has been assessed in various cellular and in vivo assays and the results have confirmed the estimated cell partition and brain to plasma ratio. It can be demonstrated, that up to 21 amino acids, the peaks of the peptides obtained on the protein phase were symmetrical and narrow. The interaction of larger peptides with the protein stationary phases resulted in wide peaks showing multiple equilibrium processes with slow kinetics during chromatography. The larger peptides showed narrow and symmetrical peaks on the IAM column enabling the quantification of peptide - cell membrane interactions.

Screening of a highly effective fluorescent derivatization reagent for carbonyl compounds and its application in HPLC with fluorescence detection

Carbonyl compounds are widely distributed in organisms, and the commonly used methods for determination of them like UV/fluorescence/mass spectrometry always require derivatization reagents. However, the reported derivatization reagents have significant difference in reactivity, which is very unfavorable for developing highly reactive reagent. In this study, we theoretically investigated the factors affecting the reactivity of hydrazine-based derivatization reagents, and proposed a strategy for filtering highly reactive reagents by quantum chemical calculation. With this strategy, N-propyl-4-hydrazino-1,8-naphthalimide (NPHNA) was filtered out as a fluorescent derivatization reagent. Taking aliphatic aldehydes as representatives, we evaluated the reactivity of NPHNA for carbonyl compounds. The derivatization of NPHNA with aliphatic aldehydes could be finished at room temperature within 60 min or 35 °C within 35 min, which showed higher reactivity than the most popular UV/MS reagent, 2,4-dinitrophenylhydrazine (DNPH). We believe that the strategy we proposed in this work is of great potential to design highly reactive UV/fluorescent/MS labeling reagents for carbonyl compounds and even other analytes.

Synthesis of penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) microsphere and its HPLC application in protein separation

In the present study, the narrow-dispersed penetrable poly(methacrylic acid-co-ethylene glycol dimethacrylate) (poly(MAA-co-EDMA)) microspheres were successfully synthesized based on the sacrificial support method. The poly(MAA-co-EDMA) microspheres mirrored the porous structure of the sacrificial support, i.e. penetrable silica, characteristic of copious mesopores and throughpores. In addition, they possessed large surface area, adjustable hydrophobicity and the cation-exchange ability. Owing to their multi functionalities, they were applied as chromatographic stationary phase to separate proteins in different separation modes, including reversed phase, hydrophobic interaction and weak cation exchange. Moreover, thanks to their throughpores, fast separation at low column backpressure could be achieved in these three modes. Both protein recovery and column stability were satisfactory. The penetrable poly(MAA-co-EDMA) microspheres were potential stationary phase matrix for fast protein separation.

The flow-through silica as the matrix to immobilize gold nanoparticles for HPLC applications

ABSTRACTIn the present study, the flow-through silica, featured with hierarchical pores, i.e., tunable mesopores and penetrable macropores, was attempted as the chromatographic stationary phase matrix to immobilize gold nanoparticles (AuNPs). It was first modified by mercapto groups (named as SiO2-SH), and then by AuNPs (named as SiO2-S-Au). Thanks to the characteristic macropores, the column backpressure of SiO2-S-Au was comparable to SiO2-SH, which effectively overcame the difficulty of high column backpressure upon the nanoparticles were introduced to the chromatographic matrix. Both the reversed-phase and hydrophilic interaction liquid chromatographic performance were observed on these two columns but with different selectivities. Hydrophobic, hydrophilic, hydrogen bond and electrostatic interactions between the SiO2-S-Au stationary phase and analytes could contribute to the retention. The SiO2-S-Au column showed excellent aqueous compatibility by “Stop-flow” test with the relative standard deviations (RSD) of analyte’s k (capacity factor) values from 0.59% to 2.88%. The reproducibility of SiO2-S-Au was acceptable with RSDs of analyte’s k values in the range of 3.13%-5.03%. In addition, compared with the SiO2-SH column, the SiO2-S-Au column had better separation performance and selectivity. The results demonstrated that the flow-through silica was a promising matrix for nanoparticles with low backpressure and different selectivities.

Dissolved organic matter, nutrients, and bacteria in Antarctic soil core from Schirmacher Oasis

Purpose This study focuses on the application of HPLC in dissolved organic matter (DOM) research in Antarctic environment together with nutrients and heterotrophic bacteria (HB) analyses. The specific aims were to investigate changes in DOM components characteristics and in nutrients in soil core from ground active layer and upper permafrost, to relate obtained data to active heterotrophic bacteria records after applying statistical data treatment methods, and to explore the potential impact of environment.

Application of high-speed counter-current chromatography and HPLC to separate and purify of three polyacetylenes from Platycodon grandiflorum.

Three polyacetylenes were isolated and purified from Platycodon grandiflorum A. DC for the first time by high-speed counter-current chromatography using a two-phase solvent system composed of hexane/ethyl acetate/methanol/water (1:31:1:31, v/v/v/v) and high-performance liquid chromatography with an Agilent ZORBAX® SB-C18 column (4.6mm × 150mm, 5 μm). After separation by high-speed counter-current chromatography and high-performance liquid chromatography, we obtained 3.5mg of platetyolin A, 4.1mg of platetyolin B, and 18.1mg of lobetyolin with purities of 97.2, 96.7, and 96.9%, respectively. The purity of each compound was assessed by high-performance liquid chromatography and the chemical structures were evaluated by high-resolution electrospray ionization time-of-flight mass spectrometry and one- and two-dimensional NMR spectroscopy. Among the isolated compounds, platetyolin A and platetyolin B are newly reported compounds.

A METHOD FOR DETERMINING 1,4-BENZOTHIAZINE DERIVATIVES IN RAT PLASMA BY HPLC AND ITS APPLICATION TO A PHARMACOKINETIC STUDY

Objective: The objective of the study was to develop, optimize and validate of a new reverse-phase high-performance liquid chromatography (RP-HPLC) method for the determining 1,4-benzothiazine derivatives (AR13 and AR15) in a biological sample of rat plasma. The 1,4-benzothiazine derivatives are produced by the synthetic reactions.Methods: RP-HPLC separation was performed using an ODS-2 Hypersil column with gradient elution mobile phase consisting of water-acetonitrile for AR13 and AR15 (1:9 v/v, 3:7 v/v) at room temperature 1 ml/min flow rate, and interfaced with photodiode array detector (PDA) detector, 233 nm, 235 nm respectively.Results: A linear response was obtained between (range from 0.100-10.00 mg/ml) AR13 and (range from 0.096–9.88 mg/ml) AR15 with correlation coefficient 0.999 and 0.998. The linearity range of both AR13 and AR15 was 101.65±1.5 and 98.78±1.7.Conclusion: It was concluded that the method was simple, accurate, sensitive, accurate and reproducible and has been successfully applied to the pharmacokinetic study of AR13 and AR15 in rat plasma.

Studies on the applicability of HPLC-FLD and HPLC–MS/MS for the determination of ergot alkaloids in rye-containing breads

The aim of this study was to elucidate the applicability of different analytical methods for the quantitation of ergot alkaloids in rye-containing bread by means of a multi-laboratory study, and to clarify how processing of rye flour into bread affects the content of ergot alkaloids at different processing stages. Wholegrain and refined rye flours were spiked to an ergot alkaloid concentration of 500 µg/kg with ergot powder and then processed into mixed rye/wheat and wholegrain rye breads. Twelve ergot alkaloids were quantitated in flour, dough, and bread by means of liquid chromatography–fluorescence detection and liquid chromatography-tandem mass spectrometry. The effect of sample drying on the ergot alkaloid concentration was also studied. The study was carried out by two laboratories; selected bread samples were additionally analysed by two further laboratories. Both laboratories and analytical methods yielded reliable results with standard deviations of 8–9 % with spiked rye flours. Short-time dough fermentation had no effect on the recoveries. Independent of the analytical method, the bread recoveries were between 42 and 109 %, showing high measurement uncertainty of up to 40 %, which was due to both a non-standardised analytical method and the calculation of the total ergot alkaloid content from 12 individual alkaloid concentrations. Air- or freeze-drying of samples prior to analysis led to partly low recoveries. In conclusion, the study showed that there is an urgent need for a standardised analytical method for the quantitation of ergot alkaloids content in baked goods to enable the reliable control of legal thresholds.

Determination of 5-hydroxymethyl-2′-deoxycytidine in Rice by High-performance Liquid Chromatography–Tandem Mass Spectrometry with Isotope Dilution

ABSTRACT5-hydroxymethyl-2′-deoxycytidine (5-hmdC), which plays important roles in the epigenetic remodeling in mammals, has been recently determined in plant genomes at trace concentrations. In this study, a method for the sensitive, specific, and rapid assessment of 5-hmdC in rice is reported using high-performance liquid chromatography–tandem mass spectrometry (HPLC–tandem MS) with isotope dilution. The recovery of this method was from 75.6% to 81.3% for 5-hmdC and the intra- and inter-day precision values were within 5.81%. The quantification limit for 5-hmdC was 5.0 pM. Genomic DNA from rice was extracted and enzymatically digested for nucleotides for the HPLC–tandem MS assay. The 5-hmdC levels were 1.32 ± 0.08, 1.64 ± 0.12, and 1.98 ± 0.07 modifications per million nucleosides in the Shengdao-18, Shengxiang-145, and Jindao-263 rice cultivars. The application of HPLC–tandem MS for 5-hmdC demonstrated its presence in the genomic DNA of rice and is useful in further studying the role of modified cytosine in the epigenetic regulation of rice.

Application of HPLC and response surface methodology for simultaneous determination of curcumin and desmethoxy curcumin in Curcuma syrup formulation

Liquid Chromatography and Fourier Transform Infrared Spectroscopy for quantitative analysis of individual and total curcuminoid in Curcuma longa extractRatna Wulandari, Sudjadi, Sudibyo Martono, Abdul Rohman

Synthesis of Esters and Carbamothioates Contaminating Tramadol Moiety and Their HPLC Applications

Synthesis of Esters and Carbamothioates Contaminating Tramadol Moiety and Their HPLC Applications

HPLC method validation and application for organic acid analysis in wine after solid-phase extraction

<p>A solid-phase extraction method followed by reverse phase high-performance liquid chromatography (RP-HPLC) was optimized and validated for the quantitative determination of tartaric, malic, shikimic, lactic, citric and succinic acids in wine. Solid-phase extraction was carried out with C18 cartridges and extraction recoveries for all acids ranging from 98.3 to 103% were obtained. HPLC separation was performed with isocratic elution on a LiChrosorb RP-18 column (250 × 4.6 mm I.D., 5 µm) protected with the appropriate guard column. The mobile phase was a 5 mM solution of H<sub>3</sub>PO<sub>4</sub> with pH 2.1 at a flow rate of 1 ml/min. Detection of the organic acids was performed at 210 nm. The developed method was validated by checking its linearity, limit of detection (LOD), limit of quantification (LOQ), precision and recovery. The method was applied to the analysis of organic acids in Macedonian red and white wines.</p>

Application of HPLC to Investigate the Physicochemical Properties and Intestinal Permeability of Ketoprofen

Application of HPLC to Investigate the Physicochemical Properties and Intestinal Permeability of Ketoprofen

Application of HPLC for the screening of separation of new macrocyclic systems

GRAPHICAL ABSTRACT

High-Throughput, High-Resolution Peptide Maps

Genetic Engineering & Biotechnology NewsVol. 36, No. 12 Bioprocessing Tech NoteHigh-Throughput, High-Resolution Peptide MapsRowan Moore and Martin SamonigRowan MooreRowan Moore (E-mail Address: rowan.moore@thermofisher.com) is vertical marketing manager for pharmaceutical and biopharmaceutical HPLC and IC and Martin Samonig is a biopharmaceutical HPLC and MS applications scientist at Thermo Fisher Scientific. Website: www.thermofisher.com/builtforbiopharma.Search for more papers by this author and Martin SamonigSearch for more papers by this authorPublished Online:8 Jun 2016https://doi.org/10.1089/gen.36.12.11AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetailsCited byStructural and compositional diversity of fibrillin microfibrils in human tissues16 February 2018 | Journal of Biological Chemistry, Vol. 293, No. 14 Volume 36Issue 12Jun 2016 InformationCopyright © GEN PublishingTo cite this article:Rowan Moore and Martin Samonig.High-Throughput, High-Resolution Peptide Maps.Genetic Engineering & Biotechnology News.Jun 2016.20-21.http://doi.org/10.1089/gen.36.12.11Published in Volume: 36 Issue 12: June 8, 2016PDF download

Just-Dip-It (Potentiometric Ion-Selective Electrode): An Innovative Way of Greening Analytical Chemistry

“Green analytical chemistry” (GAC) is a vital area towards the concept of sustainability. As a consequence of the widespread application of HPLC in drug-related analytical investigations and the resulting contamination of the environment with organic solvents questions have been raised about the toxicity/greenness of HPLC in the ecosystem. Traditional analytical separation technologies yield approximately 50 mL of waste per analytical data point. To this end, the pharmaceutical community continues to search for greener opportunities to markedly reduce the amount of organic waste produced and move from conventional offline separation based methodologies to greener in-line alternatives. In this contribution, we’re adopting a “Just-Dip-It” approach with the ultimate goal of advancing and exploiting the potentiometric sensors to their most effective use in different disciplines of drug development. The unique abilities of these ion-selective electrodes (ISEs) for in-line measurements is the key driver for ado...

Simultaneous Sensitive Determination of Eight Collagen-Related Bone Turnover Biomarkers in Diverse Clinical Samples by HPLC with Fluorescent Derivatization

The suitability of HPLC with fluorescent derivatization for simultaneous determination of eight collagen-related bone turnover biomarkers, 4-hydroxy-l-proline (OHP), pyridinoline (PYD), hydroxylysine (Hyl), deoxypyridinoline, and their glycosides, galactosyl-Pyd (Gal-Pyd, GP), galactosyl-glucosyl-Pyd, β-1-galactosyl-O-hydroxylysine (Gal-Hyl, GH), and α-1,2-glucosyl-galactosyl-O-hydroxylysine (GGH), in diverse clinical samples was investigated. 5-Carboxyfluorescein succinimidyl ester (CFSE) was chosen as the fluorescent labeling reagent. Various parameters affecting derivatization and separation were optimized. Under optimal conditions, maximum derivatization could be achieved in 25 min at 30 °C, and complete baseline separation could be completed within 25 min, with relative standard deviation values for corrected peak areas less than 3.5 % for intraday assay and 4.2 % for interday assay, respectively. The limits of detection were determined to be 0.1–1.7 nM, which are well below the expected concentrations in the real samples. The method has been used for the quantitative determination of eight target analytes in various clinical samples, including cell cultures of normal human osteoclast, preosteoblast, osteoclastoma cells and serum and urine fluids from patients with fracture, osteitis fibrosa, or osteoclastoma as well as healthy subjects, with spiked recoveries of 96–103 %. This original application of HPLC–fluorescence detection represents a powerful tool for investigating the dynamics of metabolic imbalance of these biomarkers for bone turnover under physiological and pathophysiological conditions.

Application of HPLC to assess the compatibility of bisoprolol fumarate with selected excipients in mixtures by isothermal stress testing

In the present study, the compatibility between bisoprolol fumarate and selected excipients (ascorbic acid, citric acid anhydrous, butylated hydroxyanisole, polyvinylpyrrolidone, glycerol, mannitol and sorbitol) in mixtures (1:10 ratio of drug and excipient) was investigated by subjecting the samples to isothermal stress conditions (90 °C for 48 h). A new HPLC method was developed, validated and employed for determining the drug content of the stressed compatibility samples. Results of HPLC revealed that major degradation of bisoprolol fumarate was observed with butylated hydroxyanisole (89.4%), citric acid anhydrous (89%), mannitol (77%) and glycerol (61.9%).

Application of HPLC–DAD after SPE/QuEChERS with ZrO2-based sorbent in d-SPE clean-up step for pesticide analysis in edible oils

In this study, the solid-phase extraction/quick, easy, cheap, effective, rugged and safe (SPE/QuEChERS) technique was adapted to develop a simple sample treatment for multi-residue pesticide analysis of edible oils. The proposed method is based on liquid–liquid partitioning with acetonitrile followed by dispersive solid phase extraction using zirconia-coated silica particles for extract purification. To evaluate the described method, 21 pesticides belonging to different chemical classes were analysed using high performance liquid chromatography with diode-array detection (HPLC–DAD). For validation purposes, recovery studies were performed at 75ngg−1, 125ngg−1, 250ngg−1, 500ngg−1 and 1000ngg−1 levels. Recoveries were over the range of 50–130% for most of the analytes, with relative standard deviations less than 15% being observed. HPLC–DAD provided suitable linearity, precision and accuracy. The validated method was successfully applied to the analysis of edible oil samples selected from the market.

Pyrrolizidine alkaloids in herbal teas for infants, pregnant or lactating women

A general contamination of tea with pyrrolizidine alkaloids (PA) has just become known. Here, we report the application and modification of a new HPLC–ESI-MS/MS sum parameter method to quantitate PA content of herbal teas intended for infants, pregnant and lactating women. Using p-toluenesulfonyl isocyanate for derivatization and a stable isotope labeled internal standard, the total retronecine-/heliotridine-type PA content of the samples is expressed in form of a single sum parameter (retronecine equivalents: RE). The new methods were applied to analyze 44 tea samples for such consumer groups. Thirty eight products (86%) were tested PA positive showing PA concentrations ranging from 0 to 391μg RE/kg (average: 50μg RE/kg). The dataset is discussed in the view of the current discussion on PA in the food chain with special focus on those particular vulnerable consumer groups.