Reversed-phase High-performance Liquid Chromatography Conditions Research Articles

Comparative analysis of trilobal capillary-channeled polymer fiber columns with superficially porous and monolithic phases toward reversed-phase protein separations.

A trilobal capillary-channeled polymer fiber stationary phase is evaluated for its performance for intact protein separations under reversed-phase high-performance liquid chromatography conditions. The separation quality, operational characteristics, and protein dynamic loading capacity on the fiber phases are compared to commercially-available superficially porous and monolithic columns. The trilobal or "y-shaped" polypropylene fiber phase was employed to separate a synthetic mixture of five proteins (having diverse chemistries and molecular weights). The separation quality was evaluated based on the resolution, peak heights/recoveries, peak widths, and peak areas. The present work illustrates the unique ability to operate at higher linear velocities (47.5mm/s) while maintaining lower back pressures (∼4 MPa), faster separation times (<8 min), and faster gradient rates using the fiber columns while yielding comparable chromatographic performance to the commercial columns. The separations employing the commercial stationary phases operate at lower linear velocities (∼3.0 mm/s), higher back pressures (∼9 MPa), require longer separation times (10 min), and require slightly higher compositions of organic mobile phase to effect protein elution. Likewise, based on breakthrough loading analysis of lysozyme and bovine serum albumin, the trilobal, polypropylene C-CP fiber column stationary phases demonstrate 3-9X greater binding capacities on a bed volume basis versus the commercial columns.

Method development on reverse-phase high-performance liquid chromatography for the improvement of routine analysis of flavonoids on agricultural and food products

Objectives: The objective of this work was to investigate the effect of reversed-phase high-performance liquid chromatography conditions for the determination of flavonoids in the plant extract. Methods: The analytical conditions (total flow rate, mobile phase, colum temperature, and detection wavelength) optimized for gallic acid, rutin, and quercetin. The optimized method was compared with the control method and evaluated with the use of reference compounds. Results: The result shows that the maximum flavonoids recovery was in observed on modified chromatographic conditions, Colum: Octadecyl-silica C18column (250 mm × 4 mm, 5 μm) Mobile Phase solution water-acetic acid (25:1) and methanol. Mobile Phase solution B was a Mobile phase – 4th min - 20%, 10th min - 80%, and a flow rate of 0.5 mL/min. Colum temperature 35°C and detection wavelength was 260 nm. Conclusion: In this work, the proposed method will increase the analysis time but minimize the mobile solvents and have the potential of being useful for the routine analysis of flavonoids.

Design of experiment avenue for development and validation of RP-HPLC-PDA method for determination of apremilast in bulk and in in-house tablet formulation

BackgroundApremilast is phosphodiesterase-4 and an immunomodulating agent used for treatment of refractory psoriatic arthritis.MethodsThe reversed-phase high-performance liquid-chromatography method for analysis of apremilast was developed and validated as per ICH guidelines. The separation of apremilast was performed on PrincetonSPHERE Ultima C18 column (250 mm × 4.6 mm, i.d., 5 μm particle size) with photodiode array detection carried out at 231 nm. A Box–Behnken design with response surface methodology was executed out for optimization of chromatographic conditions of reversed-phase high-performance liquid-chromatography for finished desired chromatographic separation of apremilast from its formulation with less number of experimental trials. Three independent factors, namely methanol composition in the mobile phase, pH of an aqueous phase, and flow rate, were used to construct a mathematical model and study the effects of these independent factors on responses such as retention time, theoretical plates, and tailing factor.ResultsOptimized experimental conditions for proposed work consists of methanol and water, pH 3.50 adjusted with ortho-phosphoric acid (70:30 % v/v) as a mobile phase at a flow rate 1 ml/min with a retention time was found to be 5.15 min. Accuracy study was completed at three different levels and was found in the range of 99.44–101.49%.ConclusionThe 3D response surface graphs revealed that the methanol composition and pH of an aqueous phase were both most stringent factors affecting the responses. Thus, a new, precise, and accurate HPLC method was developed and validated and can be used for regular analysis of apremilast.

Establishment and Application of Infant Formula Fingerprints by RP-HPLC

An efficient method for rapid and simple identification of infant formula quality was investigated so as to prevent brand counterfeit of infant formulas. Reversed-phase, high-performance liquid chromatography (RP-HPLC) method combined with chemometric method was developed for fingerprints analysis. To optimize the RP-HPLC conditions, the RP-HPLC analysis was performed on a C8 column by using gradient elution with 0.1% (v/v) aqueous trifluoroacetic acid (TFA) in water as mobile phase A and 0.1% (v/v) TFA in acetonitrile as mobile phase B, the flow rate was 0.5 mL min−1, and the detection wavelength was set at 214 nm. Three different stages of infant formulas of two series (R1 and R2) of R brand were analyzed to establish infant formula fingerprints of each series at different stages under optimal conditions. Five major common peaks in each chromatogram of the infant formulas were used in fingerprint analysis. The results showed that the RP-HPLC fingerprints and principal component analysis (PCA) method can effectively differentiate the infant formula of different brands, as well as the different series of infant formulas of the same brand, which provide an effective testing method for authenticity identification and brand protection in infant formulas.

Effect of the topology of benzotriazole derivatives on their chromatographic retention under RP-HPLC conditions

Sixth order connectivity indices are calculated for 29 benzotriazole derivatives. Correlations between the connectivity indices, physicochemical characteristics, and retention factors of the mentioned compounds are obtained for reversed-phase high-performance liquid chromatography conditions. Based on the correlations obtained, a predictive calculation of the retention factors of some benzotriazole derivatives is performed.

New potential humic acid stationary phase toward drug components: Development of a chemometric-assisted RP-HPLC method for the determination of paracetamol and caffeine in tablet formulations.

A new humic acid based stationary phase has been used, for the first time, to achieve the separation and quantification of paracetamol and caffeine in pharmaceutical preparations under reversed-phase high-performance liquid chromatography conditions. Central composite design was applied as a powerful tool to optimize the most dominant parameters that influence the resolution of reversed-phase high-performance liquid chromatography, that is, mobile phase composition (acetonitrile percentage in water), flow rate, and column temperature. The optimum conditions were obtained as 21.69%, 1.5 mL/min, and 15°C, respectively, with the aid of a second-order quadratic model and desirability function. Under the optimum conditions, the peaks could be baseline separated within 10 min. For the developed reversed-phase high-performance liquid chromatography method, the linearity was investigated in the concentration ranges of 2-160 mg/mL (R(2) = 0.999) for paracetamol and 2-9.9 mg/mL (R(2) = 0.991) for caffeine. Mean recoveries for paracetamol and caffeine were 95.90 and 95.68%, respectively. The limits of detection and quantification were 4.1 × 10(-4) and 1.3 × 10(-3) mg/mL for paracetamol and 1.6 × 10(-4) and 5.0 × 10(-4) mg/mL for caffeine. The results showed that the new humic acid based stationary phase is very suitable for the separation of paracetamol and caffeine in pharmaceutical preparations and, thus it can be used effectively in the pharmaceutical industry.

Separation of Tegafur and its Impurities by Reversed-Phase High-Perfomance Liquid Chromatography

Abstract The chromatographic behavior of tegafur and its impurities on a naphthalene Cosmosil piNAP column under reversed-phase high-performance liquid chromatography conditions was examined. A good description of the retention was achieved through the application of statistical weights to the widely used quadratic relationships between the logarithm of the retention factor (log k) and the organic solvent concentration in the mobile phase. Optimum conditions for isocratic separation of the compounds were found with acetonitrile concentration of 10-30% in the mobile phase

HPLC Retention Thermodynamics of Grape and Wine Tannins

The effect of grape and wine tannin structure on retention thermodynamics under reversed-phase high-performance liquid chromatography conditions on a polystyrene divinylbenzene column was investigated. On the basis of retention response to temperature, an alternative retention factor was developed to approximate the combined temperature response of the complex, unresolvable tannin mixture. This alternative retention factor was based upon relative tannin peak areas separated by an abrupt change in solvent gradient. Using this alternative retention factor, retention thermodynamics were calculated. Van't Hoff relationships of the natural log of the alternative retention factor against temperature followed Kirchoff's relationship. An inverse quadratic equation was fit to the data, and from this the thermodynamic parameters for tannin retention were calculated. All tannin fractions exhibited exothermic, spontaneous interaction, with enthalpy-entropy compensation observed. Normalizing for tannin size, distinct tannin compositional effects on thermodynamic parameters were observed. The results of this study indicate that HPLC can be valuable for measuring the thermodynamics of tannin interaction with a hydrophobic surface and provides a potentially valuable alternative to calorimetry. Furthermore, the information gathered may provide insight into understanding red wine astringency quality.

Protein Covalent Dimer Formation Induced by Reversed-Phase HPLC Conditions

Reversed-phase high-performance liquid chromatography (RP-HPLC), which is routinely used to detect and quantitate levels of protein oxidation, was used to analyze a free cysteine-containing protein. However, the RP-HPLC method appeared to induce dimerization of the oxidized protein. The purpose of this study was to understand the role of RP-HPLC conditions in inducing protein dimerization. Samples were also analyzed by orthogonal size-based analytical methods such as size-exclusion high-performance liquid chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis. These methods indicated the presence of dimer and confirmed that the acidic solvent conditions induced the dimer formation of the oxidized protein. Furthermore, the dimerization was observed only when the protein was mildly oxidized and not when the protein was severely oxidized or in its native form. The sulfenic acid form of cysteine is a likely precursor to the disulfide formation. The amount of dimers increased with increasing concentration of trifluoroacetic acid (TFA) or formic acid is in the range of 0%-0.3%. The effect of the organic solvent was less than the effect of TFA/formic acid on dimer formation. Given that RP-HPLC is typically run with low-pH mobile phase containing an ion-pairing acid for improved resolution, its potential for inducing artifacts needs to be taken into consideration during method development.

Simultaneous Determination of Glutamate, Glycine, and Alanine in Human Plasma Using Precolumn Derivatization with 6-Aminoquinolyl-N-hydroxysuccinimidyl Carbamate and High-Performance Liquid Chromatography

A simple, sensitive and reproducible high-performance liquid chromatography (HPLC) method has been validated for determining concentrations of glutamate, glycine, and alanine in human plasma. Proteins in plasma were precipitated with perchloric acid, followed by derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Simultaneous analysis of glutamate, glycine, and alanine is achieved using reversed-phase HPLC conditions and ultraviolet detection. Excellent linearity was observed for these three amino acids over their concentration ranges with correlation coefficients (r)>0.999. The intra- and inter-day precision were below 10%. This method utilizes quality control samples and demonstrates excellent plasma recovery and accuracy. The developed method has been successfully applied to measure plasma glutamate, glycine, and alanine in twenty volunteers.

Analysis of human luteinizing hormone and human chorionic gonadotropin preparations of different origins by reversed-phase high-performance liquid chromatography

Specific reversed-phase high-performance liquid chromatography conditions are reported for the analysis of recombinant and native human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) preparations. Heterodimeric hLH, hCG and their α- and β-subunits migrated with significantly different retention times ( t R) in the following order of increasing hydrophobicity: α-hCG < α-hLH < hCG < hLH < β-hCG < β-hLH. Under these conditions, the main peak of three hCG preparations ran about 4% faster than the average t R (38.35 ± 0.42 min; RSD = 1.1%) of four hLH preparations. Four heterogeneous urinary products were also analyzed, hLH, hFSH and hCG peaks being identified. Quantitative analysis was validated for the homogeneous preparations and a highly linear dose–response curve ( r = 0.99998; p < 0.0001; n = 20) used to assess the accuracy, precision and sensitivity of the analysis. Quantification of the different gonadotropins in the heterogeneous preparations was also carried out, but with limitations in accuracy.

Sensitive and selective liquid chromatography/tandem mass spectrometry methods for quantitative analysis of 1-methyl-4-phenyl pyridinium (MPP +) in mouse striatal tissue

The systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to mice produces a reliable and selective degeneration of the nigrostriatal pathway, a hallmark feature of Parkinson’s disease (PD). Determining the brain concentrations of 1-methyl-4-phenyl pyridium (MPP +), the neurotoxic metabolite of MPTP, is critical for evaluating drugs designed to potentially treat PD. We have developed sensitive and specific quantitative methods for the determination of MPP + in mouse striatal tissue by liquid chromatography/tandem mass spectrometry. The separations were carried out based on reversed phase chromatography or cation exchange chromatography with volatile elution buffer. Neutralizing the brain sample with 0.2 M phosphate buffer successfully solved a high-performance liquid chromatography (HPLC) peak tailing of MPP + in brain extracts with 0.4 M perchloric acid (HClO 4) under the reversed phase HPLC conditions, which significantly improved the sensitivity of the method. The HPLC peak shape of MPP + using cation exchange chromatography was not affected by the pH of the samples. Optimization of electrospray ionization (ESI) conditions for the quaternary ammonium compound MPP + established the limits of detection (LOD) (S/N = 3) at 0.34 pg/mg tissue and 0.007 pg/mg tissue (5 μl of injection) using the reversed phase liquid chromatography/tandem mass spectrometry (LC/MS/MS) and the cation exchange LC/MS/MS, respectively. Both methods were selective, precise (%R.S.D. < 6%), and sensitive over a range of 0.001–1 ng/mg tissue. The cation exchange method showed greater sensitivity and tolerance to low pH samples than the reversed phase method. The developed methods were applied to monitoring changes in MPP + concentrations in vivo. Two reference agents, R-(−) Deprenyl and MK-801, known to alter the concentration of MPP + in MPTP treated mice were evaluated.

Interaction of native α-cyclodextrin, β-cyclodextrin and γ-cyclodextrin and their hydroxypropyl derivatives with selected organic low molecular mass compounds at elevated and subambient temperature under RP-HPLC conditions

The main focus of this study was to explore the capability of native alpha-cyclodextrin, beta-cyclodextrin and gamma-cyclodextrin and their hydroxypropyl derivatives for host-guest interaction with 7,8-dimethoxyflavone, selected steroids (estetrol, estriol, estradiol, estrone, testosterone, cortisone, hydrocortisone, progesterone and 17alpha-hydroxyprogesterone) and polycyclic aromatic hydrocarbons (toluene, naphthalene, 1,8-dimethylnaphthalene, 1-acenaphthenol, acenaphthylene and acenaphthene) under reversed-phase liquid-chromatography conditions. The study revealed that native cyclodextrins interact more efficiently with the analytes investigated than do their hydroxypropyl counterparts. In the low-temperature region, enormously high ratios were observed for polycyclic aromatic hydrocarbons, particularly 1,8-dimethylnaphthalene, acenaphthene and acenaphthylene chromatographed on a beta-cyclodextrin-modified mobile phase. In such a case, the retention times of the polycyclic aromatic hydrocarbons were strongly reduced (e.g. from 127 to 1.2 min for 1,8-dimethylnaphthalene) and were close to the hold-up time of the high-performance liquid chromatography (HPLC) system (0.7 min). Moreover, chiral separation of 1-acenaphthenol optical isomers was observed and the elution order of the enantiomers was determined. Within the steroids group, strong interaction was observed for estradiol and testosterone. The results of cluster analysis indicate that beta-cyclodextrin as well as gamma-cyclodextrin and its hydroxypropyl derivative can be most effective mobile-phase additives under reversed-phase HPLC conditions for 3D-shape-recognition-driven separation, performed at subambient and elevated temperatures, respectively.

Simultaneous determination of total homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma by high-performance liquid chromatography: Application to studies of oxidative stress

A sensitive, reproducible, and robust high-performance liquid chromatography (HPLC) method has been validated for simultaneously determining total concentrations of the aminothiols homocysteine, cysteine, cysteinylglycine, and glutathione in human plasma. Plasma aminothiols are reduced via incubation with tris-(2-carboxyethyl)-phosphine hydrochloride, followed by protein precipitation with trichloroacetic acid and derivatization with ammonium-7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonic acid. Separation of aminothiols and the internal standard mercaptopropionylglycine is achieved using reversed-phase HPLC conditions and fluorescence detection. Excellent linearity is observed for all analytes over their respective concentration ranges with correlation coefficients ( r) > 0.99. The intra- and inter-day precision and accuracy were within ±10%. This method utilizes an internal standard, employs phosphate buffered saline-based standards and quality controls, and demonstrates excellent plasma recovery and improved sensitivity. This assay is well suited for high-throughput quantitative determination of aminothiols in clinical studies, and is currently being used to support investigations of oxidative stress in patients with chronic kidney disease.

Application of Alkylamide Phase to the Separation of Inorganic Anions by Reversed Phase HPLC

Packing with an alkylamide chemically bonded stationary phase (AP) for high performance liquid chromatography (HPLC) was applied for the determination of inorganic anions in water samples. The conventional octadecyl (C18) stationary phase was used as a reference column packing material for reversed‐phase (RP) HPLC. The mobile phase composition under ion‐pair RP HPLC conditions, was optimized by changing the concentration of the organic modifier (acetonitrile), the ionic strength of eluent (addition of TRIS), and the pH value. The data obtained were used for qualitative and quantitative determination of anions in samples of drinking water and water from the Vistula River.

LC/NMR in Natural Products Chemistry

Efficient detection and rapid characterisation of natural products play an important role for their study in complex biological matrices such as crude plant extracts. Their preliminary identification at the earliest stage of separation is a strategic element for guiding selective isolation procedures. Hyphenated techniques such as high performance liquid chromatography (HPLC) coupled to UV and mass spectrometry (LC/UV/MS) have proved to be extremely useful in combination with biological screening for a rapid survey of natural products. However, these coupled techniques do not allow a full on-line identification, except for some well-known natural products, and complementary on-line spectroscopic information is needed. Thus, HPLC coupled to nuclear magnetic resonance (LC/NMR) represents a potentially interesting complementary technique to LC/UV/MS for detailed on-line structural analysis. Indeed recent progress in NMR technology has given a new impulse to LC/NMR which is now emerging as a powerful analytical tool. The development of efficient solvent suppression techniques enables the measurement of high quality LC/&lt;sup&gt;1&lt;/sup&gt;H-NMR spectra, both on-flow and stop-flow, with reversed-phase HPLC conditions. Non-deuterated solvents such as MeOH or MeCN can be used, while water is replaced by D&lt;sub&gt;2&lt;/sub&gt;O. In this review, a summary of the basic principles and modes of operation of LC/NMR are briefly described. Selected examples of LC/NMR analyses of natural products in crude p lant extract o r in enriched fractions are presented. They include different ways of using this technique. An overview of the relatively scarce applications with natural products is given. The potential and the limitations of LC/NMR in conjunction with LC/UV/MS for the on-line analysis of plant metabolites are discussed.

Lability of zinc dialkyldithiophosphates under reversed-phase high-performance liquid chromatography conditions

Investigations of the behaviour of zinc dialkyldithiophosphates in aqueous-based media indicate that the species retained in reversed-phase HPLC on a C 18 column is not the zinc complex. Electrospray mass spectrometric (ES-MS) studies on collected chromatographic peaks show no evidence for the presence of zinc in these samples in the negative and positive ion modes. Electrochemical studies support the mass spectral data by indicating that the zinc complexes dissociate in aqueous-based media to yield free ligand.

Labeling of insulin with non-radioactive 127I and application to incorporation of radioactive 125I for use in receptor-binding experiments by high-performance liquid chromatography

Conditions for the labeling of insulin with radioactive iodine isotopes were investigated by means of incorporation of non-radioactive 127I into the peptide. Either the chloramine-T (CT) or lactoperoxidase-hydrogen peroxide (LPO) technique was applied and reversed-phase high-performance liquid chromatography (RP-HPLC) was used for analysis of the reaction products. The LPO method provided the 127I-labeled peptide within 15–30 min, whereas the CT alternative yielded the labeled substrate even within 15 s. However, the latter reaction can only be controlled in a reproducible manner with difficulty and undesirad side-reactions became increasingly prominent when t a few seconds. In another experiment, the LPO technique was applied for radiolabeling insulin with 125I. The product was first purified by size-exclusion chromatography (SEC) and then subjected to RP-HPLC. SEC yielded two peaks. The smaller one, which eluted at a slightly higher K d value (accounting for about 14% of total radioactivity) predominantly consisted of material eluting at the column's void volume under the conditions of RP-HPLC, whereas the main SEC fraction (accounting for about 86% of total radioactivity) yielded a single peak, as shown by HPLC. The radioactive material attributable to the main SEC fraction revealed the expected receptor-binding properties, as evidenced by displacement experiments with non-radioactive insulin, as well as the action of tetradecanoyl phorbol acetate on the binding characteristics and thus indicating formation of a labeled hormone retaining biological activity.

Separation and characterisation of bovine histone H1 subtypes by combined ion-exchange and reversed-phase chromatography and mass spectrometry.

In order to separate and identify histone H1 subtypes from calf thymus we used both electrospray mass spectrometry (ES-MS) and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS) after a three-step chromatographic procedure consisting of reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC) and ion-exchange chromatography (IEC). Under the RP-HPLC conditions described, we obtained two baseline-separated H1-fractions which were characterised by MALDI-TOF-MS. The determined masses ranged from 22,850 to 22,590 for the first fraction and from 22,070 to 21,250 for the second fraction. Further, it was shown that the first fraction contained at least four and the second one at least five subtypes of the histone class H1. Four homogeneous pure H1 subtypes were obtained by a combination of IEC followed by SEC and RP-HPLC. The molecular masses of these four subtypes determined by ES-MS were 22,606, 22,761, 21,347 and 21,263. We obtained six additional molecular masses of histone H1 subtypes from three heterogeneous fractions, namely 22,066, 21,802, 20,586 and 19,817 by ES-MS and 22,800 and 22,675 by MALDI-TOF-MS. The retention times of these fractions and the molecular masses were in agreement with the data obtained from RP-HPLC fractions by MALDI-TOF-MS.

High-performance liquid chromatography of amino acids, peptides, and proteins. 123. Dynamics of peptides in reversed-phase high-performance liquid chromatography

The dynamics of several peptides in reversed-phase high-performance liquid chromatography (RP-HPLC) have been investigated on both n-octadecyl (C18) silica and n-butyl (C4) silica sorbents. In particular, the conformational interconversions and the relative rates of chromatographic relaxation of bombesin, glucagon, and beta-endorphin on both C18 and C4 n-alkylsilicas were monitored by examining changes in the experimental bandwidths of these peptides as a function of temperature and column residence time under linear gradient elution RP-HPLC conditions. The observed band-broadening trends were correlated with previously derived retention parameters and thermodynamic descriptors of the association process determined for bombesin, beta-endorphin, glucagon, and a control peptide, penta-L-phenylalanine. This study confirms that bandwidth measurements can be used as an integral experimental component to study the effect of the secondary structure of peptidic solutes on their RP-HPLC retention behavior. Further, the data demonstrate the utility of RP-HPLC as a tool to examine peptide conformational dynamics at hydrophobic surfaces. The relevance of these results to the general phenomenon of peptide-lipid interactions is discussed in terms of the associated evidence for lipid-induced changes in the conformation of these three bioactive peptides.