Reversed-phase HPLC Conditions Research Articles

Specific Features of the Determination of Xanthophyll Esters under Reversed-Phase HPLC Conditions

Specific features of chromatographic determination of xanthophyll esters are studied using an example of marigold flower lutein diesters under reversed-phase HPLC conditions. The developed two-column method made it possible to establish that, in samples with a low solubility of carotenoids in the used solvent and on a chromatography column in using a mobile phase with a low solubility of carotenoids, the precipitation of diesters is possible, which detrimentally affects the accuracy of the chromatographic determination. A critical factor in this case isì temperature: the storage of samples (solutions) in a refrigerator is not always advisable, because freezing of the main components is possible. It was shown that the use of a mobile phase containing from 0 to 10 vol % acetonitrile in acetone at a temperature not lower than 20°C is acceptable for the separation of all-trans-lutein diesters from cis-derivatives of lutein and zeaxanthin derivatives on “monomeric” C18-phases.

Specific Features of the Determination of Xanthophyll Esters under Reversed-Phase HPLC Conditions

Specific Features of the Determination of Xanthophyll Esters under Reversed-Phase HPLC Conditions

Chiral HPLC separation of enantiomeric blebbistatin derivatives and racemization analysis in vertebrate tissues

Simple and consistent chiral HPLC methods for the efficient separation of enantiomeric blebbistatin derivatives, namely parent compound blebbistatin and derivatives 4-nitroblebbistatin, 4-aminoblebbistatin, 4-dimethylaminoblebbistatin, and 4-t-butylblebbistatin were developed using cellulose tris(3,5-dimethylphenylcarbamate) as a stationary phase (Lux cellulose-1 column). Blebbistatin, 4-aminoblebbistatin, and 4-dimethylaminoblebbistatin racemates were well-separated in normal-phase HPLC conditions while 4-nitroblebbistatin and 4-t-butylblebbistatin were effectively separated in both normal- and reversed-phase HPLC conditions. Furthermore, the order of elution of enantiopure compounds was found to be independent of mobile phase compositions and conditions used, and solely depends on the interaction between the enantiomer and the chiral stationary phase. We found that despite the chiral center being present far from the D-ring in the blebbistatin structure, the D-ring substitutions prominently affect the chiral separation. Ex vivo racemization studies of the most popular blebbistatin derivative (S)-(–)-4-aminoblebbistatin in rat blood and brain tissues revealed that the compound does not convert into the inactive enantiomer. This confirms that (S)-(–)-4-aminoblebbistatin is a useful tool compound in cellular and molecular biology studies without the risks of racemization and degradation effects.

New composite stationary phase for chiral high-performance liquid chromatography

The new composite chiral stationary phase for high-performance liquid chromatography was prepared and characterized. Poly(styrene-divinylbenzene) microspherical particles with diameter of 3.3 micron were used as a matrix and coated with layer of 10 nm gold nanoparticles. Immobilized gold nanoparticles increased specific surface area of adsorbent and simplify covalent attachment of sulfur-containing compounds. In this work l-lysine conjugate with lipoic acid was synthesized, characterized and used for modification of gold nanoparticles. The prepared chiral selector was immobilized by the reaction of sulfur-containing groups from lipoic acid residue with gold surface of nanoparticles with formation of self-assembled monolayer. The prepared chiral stationary phase was characterized by nitrogen adsorption at low temperatures, diffuse reflection spectroscopy, scanning electron microscopy. The chromatographic retention of beta-blockers and profens was studied under conditions of reversed-phase HPLC. The possibility of enantiomers separation was demonstrated for flurbiprofen and ketoprofen racemates using 100 × 4.6 mm ID chromatographic column.

Confirmation of organic compounds hydrates formation under the reversed-phase HPLC conditions

The confirmation of the correspondence of the detected forms of analytes to their molecular structures seems to be one of the principal problems of analytical chemistry. The previous analysis of the series of drugs by reversed-phase HPLC allowed revealing that the retention regularities of the complex polyfunctional organic compounds containing sulfonamide groups -SO2-N< differ from those of other compounds. It was explained by reversible formation of their hydrate forms. To verify this proposition, three model N-substituted arylsulfon­ami­des were synthesized, namely N,N-diethyl- (I), N-allyl- (II), and N-phenyl (III). The consideration of the dependencies of these analytes net retention times (tR) in reverse-phase HPLC on the content of orga­nic solvent (C, methanol) in an eluent did not permit us to reveal any regularities or anomalies due to the non-linearity of such depen­den­cies. However, to achieve this, the recurrent appro­ximation was re­com­mended, tR(C + DC) = atR(C ) + b (*), where DC = const – con­stant varia­tions of concentration of methanol (5% in our case), а and b – coefficients calculated by Least Squares Method. If the chemical origin of the analytes at the outlet of the chromatographic column remained the same within concentration range Сmin < С < Сmax, dependencies (*) were linear with correlation coefficients R > 0.999. Yet, if additional chemical transformations of analytes took place, na­me­ly rever­sible formation of hydrates (or variations of their composition) due to the presence of water in an eluent, it led to the deviations of recurrent dependencies (*) from linea­rity. Three sulfonamides under characterization corresponded to different kinds of such deviations: non-linearity within the whole range Сmin < С < Сmax (amide I), presence of two linear dependencies instead of one (amide II), and linearity within the whole range Сmin < С < Сmax (amide III). First two cases corres­ponded to the interconver­si­on of anhydrous forms and hydrates, or (less probable) different hydrates, while the latter meant the existence of analyte in the single form (pro­ba­b­ly hydrate). Thus, the ana­ly­sis of mo­del compounds confirmed that the presence of the polar functionality -SO2-N< was just the reason of hydrate formation in water soluti­ons.

Selectivity of the Separation of Isomeric Chlorogenic Acids under the Conditions of Reversed-Phase HPLC

The selectivity of the separation of isomeric chlorogenic acids often found in natural samples (3, 4, and 5-caffeoylquinic acids, 3CQA, 4CQA, and 5CQA, respectively) under the conditions of reversed-phase chromatography on stationary phases of different brands, Kromasil (C18, C8, and C4), Symmetry C18, Reprosil-Pur C18-AQ, Nucleosil RPC18, and Diasfer C18, is studied. The results obtained favor the retention mechanism with predominant surface adsorption. The change in the selectivity of separation of a pair of 5CQA and 4CQA correlates with a change in the activity of residual silanol groups. At their low activity, the order of elution 3CQA < 5CQA < 4CQA does not depend on the mode (isocratic or gradient), the type of organic modifier, and the addition of an acid (formic, orthophosphoric) to the mobile phase. With an increase in the activity of silanol groups, the separation of the pair of 5CQA and 4CQA deteriorates up to the inversion of retention times. A Kromasil 100-5C18 column (250 × 4.6 mm) was chosen for a reliable separation of three acids in plant samples (instant coffee, juice, and “Mate” tea).

Liquid Chromatography Tandem Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy of Magnesium (II) Gluconate Solution

Magnesium gluconate is a classical pharmaceutical compound used as a source of magnesium for the prevention and treatment of hypomagnesemia. To the best of our knowledge, a robust and reliable liquid chromatography tandem mass spectrometry technique has not yet been reported for the qualitative and quantitative analysis of magnesium gluconate. This study describes the method development for the LC–ESI–MS/MS analysis of magnesium gluconate using three different reversed-phase HPLC conditions (Method I–III) with comprehensive fragmentation pattern and the structural characterization by NMR spectroscopy. The LC–MS and NMR data were found in accordance with the structure of magnesium gluconate. When magnesium gluconate was dissolved in the acetonitrile and water–methanol solutions, it exists in situ in three different forms: magnesium gluconate itself, gluconic acid, and magnesium gluconate chelate with gluconic acid by a coordinate covalent bond. Method I exhibited pseudo-molecular ion peaks with more magnesium gluconate chelates with gluconic acid, while method II showed an adduct of magnesium gluconate with the solvent along with the molecular ion peak. There was no pseudo-molecular ion peaks found in method III. Thus, method III was found to be the more accurate, robust and reliable LC–MS method for the qualitative and quantitative analysis, structural characterization, and could also be suitable for the pharmacokinetic study of magnesium gluconate. The detailed fragmentation analysis might be useful for the structural characterization of unknown divalent organometallics.

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.

Synthesis of the four stereoisomers of 2,3-epoxy-4-hydroxynonanal and their reactivity with deoxyguanosine

2,3-Epoxy-4-hydroxynonanal (EHN) is a potential product of lipid peroxidation that gives rise to genotoxic etheno adducts. We have synthesized all four stereoisomers of EHN and individually reacted them with 2'-deoxyguanosine. In addition to 1,N(2)-etheno-2'-deoxyguanosine, 12 stereoisomeric products were isolated and characterized by (1)H NMR and circular dichroism spectroscopy. The stereochemical assignments were consistent with selective NOE spectra, vicinal coupling constants, and molecular mechanics calculations. Reversed-phase HPLC conditions were developed that could separate most of the adduct mixture.

Identification and quantification of degradations in the Asp–Asp motifs of a recombinant monoclonal antibody

Two degradations of aspartate residues located in Asp–Asp motifs in the CDR3 region of a recombinant monoclonal antibody were identified and quantified after the antibody was aged in a mildly acidic buffer at elevated temperatures. The degraded sample aged at 25 °C for 1 month generated 1.8% antibody molecules that had isomerization in the aspartate residues, while the degraded sample after aging at 45 °C for 1 month contained 7% isomerization. Peptide bond cleavages at the aspartate residues were also detected and characterized. The percentage of clipped antibody molecules after 1 month of storage was 1% at 25 °C and 4.4% at 45 °C. The generated cleaved polypeptides were noncovalently attached to the intact antibody molecule and were not involved in the aggregation formation. They were not detected by native size-exclusion chromatography because of their strong non-covalent association to the rest of the antibody molecules. On the other hand, the cleaved polypeptides were dissociated and detected as fragments under denaturing conditions of reversed-phase HPLC, denaturing size-exclusion chromatography and MALDI-TOF mass spectrometry. It was demonstrated that the cleavages at the above aspartate residue sites occurred due to the aging of the sample at elevated temperatures and were not method-induced by the reversed-phase HPLC and other methods used in this study.

Exploring the importance of the relative solubility of matrix and analyte in MALDI sample preparation using HPLC.

New insight into the role of solubility in the sample preparation process for MALDI MS is reported. Reversed-phase gradient HPLC conditions were developed that enable the analysis of a broad range of analyte polarities with a single method. This HPLC method was used to establish a relative polarity scale for a series of 15 MALDI matrix materials, a set of example peptides, and a series of model polymer materials with a broad range of polarity. Examples of each polymer type within the range of 6000-10,000 were analyzed with six matrixes that cover a broad range of polarity using MALDI TOFMS. With regard to polymer signal-to-noise ratio, the matrix and polymer combinations that had a close match of HPLC retention time produced the best MALDI spectra. Conversely, the matrix and polymer combinations that have a large difference in HPLC retention time produced poor MALDI spectra. The results suggest that there is a relationship between polarity (solubility) and effective MALDI sample preparation. The relative HPLC retention time of an unknown polymer can serve as a starting point for predicting the matrix (or range of matrixes) that would be most effective.

Separation and determination of minor photosynthetic pigments by reversed-phase HPLC with minimal alteration of chlorophylls.

Reversed-phase HPLC conditions for separation of chlorophyll (Chl) a, Chl a' (the C132-epimer of Chl a), pheophytin (Pheo) a (the primary electron acceptor of photosystem (PS) II), and phylloquinone (PhQ) (the secondary electron acceptor of PS 1), have been developed. Pigment extraction conditions were optimized in terms of pigment alteration and extraction efficiency. Pigment composition analysis of light-harvesting complex II, which would not contain Chl a' nor Pheo a, showed the Chl a'/Chl a ratio of 3-4 x 10(-4) and the Pheo a/Chl a ratio of 4-5 x 10(-4), showing that the conditions developed here were sufficiently inert for Chl analysis. Preliminary analysis of thylakoid membranes with this analytical system gave the PhQ/Chl a' ratio of 0.58 +/- 0.03 (n = 4), in line with the stoichiometry of one molecule of Chl a' per PS I.

Phenylaminopropyl silica – a new specific stationary phase for high-performance liquid chromatography of phenols

The chromatographic properties of a new stationary phase, phenylaminopropyl silica (PhA-silica), containing phenylaminopropyl residues covalently bonded to the silica surface were studied. The presence of secondary amino groups, phenyl rings and alkyl linkers in the attached molecule makes it especially suitable for the separation of phenols by mixed mode retention mechanism including a combination of hydrogen-bonding, hydrophobic, electrostatic and π–π interactions with the stationary phase. The effects of mobile phase pH, ionic strength, nature and concentration of organic modifier on the retention of phenols on PhA-silica were investigated under conditions of reversed-phase HPLC. To elucidate the role of the amino group in the attached molecule in retention of phenols the selectivity of PhA-silica was compared with that obtained for phenylpropyl silica in the framework of a linear solvation energy relationship (LSER) model. The isocratic separation of phenol, and its nine methyl-, chloro- and nitro-substituted derivatives was achieved on a 150×4.6 mm I.D. chromatographic column packed with 7 μm particles of PhA-silica.

Reversed-phase high-performance liquid chromatography of 4-(2-pyridyl)-1-piperazinethiocarboxylic acid 2-[1-(pyridyl)ethylidene]hydrazide dihydrochloride (NSC 348977), a synthetic thiosemicarbazone with antitumor activity

Reversed-phase HPLC conditions for the separation of 4-(2-pyridyl)-1-piperazinethiocarboxylic acid 2-[1-(pyridyl)ethylidene]hydrazide dihydrochloride (NSC 348977, I), a synthetic thiosemicarbazone with antitumor activity, from mouse plasma have been investigated. Following denaturization and precipitation of the spiked plasma with acetonitrile, an aliquot of the supernatant was diluted with aqueous buffer and subjected to analysis on a Nova-Pak C 18 column (150×3.9 mm I.D.) by isocratic elution with 50 m M aqueous potassium phosphate buffer (pH 6.8, containing 1 m M EDTA)–acetonitrile (60:40, v/v). The column effluent was monitored for UV absorption at 310 nm. Problems identified in the sample preparation and separation of I include sensitivity to oxygen, light, non-neutral pH and the presence of metal ions. These factors were seen to adversely influence sample recovery, and attempts were made to find conditions which minimize their effects.

Relationships between the chromatographic hydrophobicity indices and solute descriptors obtained by using several reversed-phase, diol, nitrile, cyclodextrin and immobilised artificial membrane-bonded high-performance liquid chromatography columns

The solvation equation [Eq. (1)] can be applied to reversed-phase HPLC capacity factors (i.e. logSP=logk).(1)logSP=c+rR2+sπH2+aΣαH2+bΣβH2+vVxS.SP is a solute property (e.g., solubility or partition coefficient) and the explanatory variables are solute descriptors as follows: R2 is an excess molar refraction, π2H is the solute dipolarity/dipolarisability, Σα2H and Σβ2H are the solute overall or effective hydrogen-bond acidity and basicity, and Vx is the McGowan characteristic volume and c, r, s, a, b and v are constants that are characteristic of the particular mobile/stationary phase combination. Only a limited hydrophobicity range can be covered using logk values obtained by isocratic chromatography at a fixed mobile phase composition. Gradient elution is more versatile and 29 compounds were examined under 20 different reversed-phase HPLC conditions using automated fast gradient methods. Using Eq. (1)with gradient retention time (tRg) in place of logSP an excellent correlation was obtained with the solute descriptors. This provides an experimental demonstration that gradient retention times (tRg) can be treated as linear free-energy related parameters just like logk or logP values. Because gradient retention times cannot be used for inter-laboratory comparisons, they were scaled by conversion to chromatographic hydrophobicity index (CHI) values by correlation with data from a calibration set of compounds with known CHI values. By comparing the coefficients r, s, a, b and v of Eq. (1)as determined from different chromatographic systems, the relative effects of different solute properties can be revealed and thus the selectivity of HPLC columns can be predicted. It is also possible to compare chromatographic behaviour with that of other partition systems, such as octanol–water, cyclohexane–water and blood–brain barrier distributions.

Reversed-phase high-performance liquid chromatography of radioiodinated salmon calcitonins

Reversed-phase HPLC conditions for simultaneous separation of salmon calcitonin, mono- and di-radioiodinated salmon calcitonins and their tryptic digested fragments have been developed. Salmon calcitonin was radioiodinated with Na 125I by the iodo-beads method. After solid-phase extraction from the reaction mixtures using C 18 Bond Elut cartridges, mono- and di-radioiodinated salmon calcitonins were separated from each other, as well as from unlabeled salmon calcitonin, on a Bondclone 10 C 18 column (300×7.8 mm I.D.) by isocratic elution with 0.1% trifluoroacetic acid in 34% aqueous acetonitrile. The characteristics of either iodinated peptides or unlabeled salmon calcitonin were evaluated on the basis of UV absorbance (215 and 280 nm), fluorescence ( λ ex=282 nm, λ em=310 nm) and measurement of specific radioactivity by means of a flow-through radio-isotope detector. HPLC separation of a tryptic digest of iodinated salmon calcitonin fraction on a W-porex 5 C 18 300 Å column (250×4.6 mm I.D.) and subsequent amino acid analysis, led to the conclusion that radioiodination took place at the Tyr residue and not at the His moiety.

Quantitation of an orally available thrombin inhibitor in rat, monkey and human plasma and in human urine by high-performance liquid chromatography and fluorescent post-column derivatization of arginine

An assay for the quantification of plasma and urine levels of CVS 1123, an orally bioavailable thrombin inhibitor, and its desmethyl form, CVS 738, was developed to support clinical and toxicology studies. This assay uses solid-phase extraction, reversed-phase HPLC separation, and post-column fluorescent derivatization with ninhydrin. An internal standard is added to correct for recovery. In aqueous solution, the arginine aldehyde structures of CVS 1123 and CVS 738 exist in multiple forms which can be separated under standard reversed-phase HPLC conditions. HPLC conditions were optimized to give rapid interconversion of the forms on the separation time scale, and consequently a single chromatographic peak. Extraction conditions were modified for quantitative extraction of drug compounds from large volumes of human plasma. The assay was shown to be accurate and precise, with a quantification limit of 17 ng CVS 1123/ml human plasma.

30-day oral toxicity study of domoic acid in Cynomolgus monkeys: Lack of overt toxicity at doses approaching the acute toxic dose

Microcystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ-TAD (4-[2-(6,7-dimethoxy-4-methyl-3-oxo-3,4-dihydroquinoxalinyl) ethyl]-1,2,4-triazoline-3,5-dione), was used as the labeling reagent. DMEQ-TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 micrograms; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70 degrees C; amount of DMEQ-TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins-LR, -YR, and -RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean-up using ODS silica gel: column, Cosmosil 5C18-AR (150 x 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL lambda ex 370 nm, lambda em 440 nm. The detection limits of the DMEQ-TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns-Cys derivatives, which were more sensitive to CL than FL.

High‐performance liquid chromatographic separation of microcystins derivatized with a highly fluorescent dienophile

AbstractMicrocystins are potent hepatotoxins produced by cyanobacteria, and are also tumor promoters as well as potent inhibitors of the catalytic subunits of protein phosphatases 1 and 2A. In order to establish a physicochemical method for individual detection and determination of trace amounts of microcystins, we developed a derivatization method for fluorescence (FL) and chemiluminescence (CL) detection, in which a highly fluorescent dienophile, DMEQ‐TAD (4‐[2‐(6,7‐dimethoxy‐4‐methyl‐3‐oxo‐3,4‐dihydroquinoxalinyl) ethyl]‐1,2,4‐triazoline‐3,5‐dione), was used as the labeling reagent. DMEQ‐TAD reacted smoothly with the conjugated diene of the Adda moiety to give 2 stereoisomers of the adducts. As a result of the extensive experiments, the following reaction conditions were optimized for the labeling: sample amount, 10 μg; reaction solvent, DMF:acetonitrile (1:1); reaction time, 15 minutes; reaction temperature, 70°C; amount of DMEQ‐TAD used relative to that of microcystin, 80 equivalent. The resulting 6 adducts from microcystins‐LR, ‐YR, and ‐RR can be separated from one another using the following reversed phase HPLC conditions in combination with a clean‐up using ODS silica gel: column, Cosmosil 5C18‐AR (150 × 4.6 I.D. mm); mobile phase, methanol:0.05M phosphate buffer (pH 3) (1:1); flow rate, 1.0 ml/min; detection, FL λex 370 nm, λem 440 nm. The detection limits of the DMEQ‐TAD derivatives were estimated to be 100 and 500 pg for LR, and 65 and 2,500 pg for RR using FL and CL detections, respectively; and the detection behavior was different from that of the Dns‐Cys derivatives, which were more sensitive to CL than FL. Nat. Toxins 5:201–207, 1997. © 1998 Wiley‐Liss, Inc.

Retention behavior of amino acids and peptides on protoporphyrin-silica stationary phases with varying metal ion centers.

Various metalloprotoporphyrins (MProP) covalently linked to silica supports are examined as novel immobilized metal ion affinity chromatography (IMAC) stationary phases for separations of amino acids/peptides. Under reversed-phase HPLC conditions, the MProP-silicas exhibit high affinity toward L-histidine via metal-nitrogen axial ligation interactions, with an increasing degree of histidine retention highly dependent on the specific metal ion (M) in the center of the protoporphyrin (ProP) structure: Fe(III) > Ni(II) > Cu(II) > Zn (II) approximately Cd(II). Aromatic amino acids (i.e., L-trytophan and L-phenylalanine) are also retained on MProP columns through pi-pi interactions with the immobilized porphyrins, with the greatest affinity for L-trytophan observed on CuProP-silica columns. Peptides rich in L-histidine and L-tryptophan residues are selectively retained on most of the MProP-silica phases examined; however, the addition of an organic modifier and/or lowering the pH of the mobile phase can be used independently to attenuate the pi-pi and metal ion-nitrogen ligation interactions, respectively. Reproducible separations of His-Phe and trytophan releasing hormone are achieved on a FeProP-silica column even after extensive washing with 50 mM EDTA, demonstrating a fundamental advantage of the new MProP-silica over existing IMAC stationary phases, in which the metal ion is anchored weakly to the support via immobilized iminodiacetate and related ligands.