Reversed-phase Performance Liquid Chromatography Research Articles

Analytical method validation of alfuzosin hydrochloride and its organic impurities by using ultra performance liquid chromatography

A low cost ultra-high performance reverse phase liquid chromatography (RPUPLC) method has been proven for the investigation of alfuzosin hydrochloride in the existence of organic pollutants and decomposition products due to forced decomposition products. Attempts have been made to develop several methods for separating drugs from organic impurities. In addition, the best chromatographic separation is Waters Acquity HSS T3 C18, 100 mm × 2.1 mm, UV detection at particle size 1.8, 254 nm, and Perchloric acid (pH 3.5 with sodium hydroxide) with a mixture of organic solvents. This method was endorsed for specificity / selectivity, linearity / range, reproducibility, recovery, and reliability. They can be utilized for quality control during manufacturing and for assessing the stability of commercially available alfuzosin hydrochloride samples. With a gradient time of about 6.0 min and an equilibration time of about 2.0 min, it was possible to analyze more than 100 samples per day. In addition, the pH susceptibility analysis methods referred to in the United States Pharmacopeia and the European Pharmacopoeia were discussed. The Method was successfully validated as per ICH Q2R1 guideline.

Development and Validation of Novel RP-HPLC Method for the Simultaneous Determination of Remogliflozin and Vildagliptin in Bulk and in synthetic Mixture

Vildagliptin which is DPP-4 inhibitor and Remogliflozin which is SGLT2 inhibitor in single dose regimen lower blood glucose by separate, complementary mechanisms. Both are glucose dependent, accounting for the low risk of hypoglycaemia during treatment. There is no risk factors associated with this combination and moreover it is single dose regimen. The aim of the present study was to develop and validate a simple, rapid and reproducible gradient high performance reverse phase liquid chromatography method for the estimation of Remogliflozin and Vildagliptin in bulk drug sample and in synthetic mixture using Xterra® Waters C18 column (150 mm×4.6 mm, 5 µm) at 25°C with UV detection at 210 nm and for this gradient mode was used. The compounds were eluted gradiently at a flow rate of 1.0ml/min. The average retention times for Remogliflozin and Vildagliptin were 4.881 and 6.334 min, respectively. The calibration curves were linear (r2 =0.988) over the concentration range 10-200 µg/ml for Remogliflozin and 10-200 µg/ml for Vildagliptin. No spectral or chromatographic interferences from formulation excipients were found and hence it was successfully applied for the determination of Remogliflozin and Vildagliptin in bulk and in synthetic mixture. The accuracy of the proposed method was determined by recovery studies and found to be 98-101%. The proposed method was validated and results conformed to ICH parameters.

Boswellic Acid Composition of Frankincense Dietary Supplements and Correlation to Cytotoxic Efficacy against Treatment‐Resistant Triple Negative Breast Cancer Cells

Frankincense is an oleogum resin from Boswellia trees of the Burseraceae family. The oleogum resin contains 15–20 % triterpenic acids, including therapeutically active boswellic acids [1]. Extracts of oleogum resins obtained from Boswellia serrata have been traditionally used in Indian folk medicine (Ayurveda) for the treatment of various chronic inflammatory diseases such as rheumatoid arthritis. Furthermore, boswellic acids are known to possess anticancer efficacy against various human cancer types, e.g. gliomas, prostate cancer, or leukemia [2–4]. The aim of the present study was to analyze the contents of boswellic acids in dietary supplements from various Boswellia species grown in different areas and to correlate the concentrations of distinct boswellic acids to the extracts' cytotoxic efficacy against chemoresistant triple‐negative human breast cancer cells. Accordingly, a new method to quantify a large variety of pentacyclic triterpenic acids was developed. Dietary supplements from various Boswellia species Boswellia serrata, Boswellia sacra, Boswellia carterii, and others were extracted with methanol and analyzed by high performance reversed‐phase liquid chromatography coupled with tandem mass spectrometric detection (HPLC‐MS/MS). Besides, the effects of the extracts against the treatment‐resistant, triple negative human breast cancer cell line MDA‐MB‐231 were investigated in vitro by using a cell viability assay. The extracts exhibited potent cytotoxic activity against the human breast cancer cell line. Correlations between the boswellic acid contents and the cytotoxic efficacy were determined by multiple linear regression and principal component regression. This approach allowed not only the correlation of complex phytochemical mixtures to desired biomedical effects, but it likewise provided evidence for key compounds governing the therapeutic efficacy. Hence, the study provides new insights into differential chemical composition patterns of frankincense dietary supplements from Boswellia species grown in different regions. Besides, the study identified distinct components correlating positively with the desired cytotoxic activity. Thus, the identified key compounds deserve further studies either as chemically pure substances or as defined chemical mixtures permitting development of the most active ingredients as potential anticancer drug candidates.Support or Funding InformationThis work was supported by the Academic Center for Complementary and Integrative Medicine (AZKIM), State Ministry of Baden‐Württemberg for Sciences, Research and Arts.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Robust naphthyl methacrylate monolithic column for high performance liquid chromatography of a wide range of solutes

An organic monolithic column based on the co-polymerization of 2-naphthyl methacrylate (NAPM) as the functional monomer and trimethylolpropane trimethacrylate (TRIM) as the crosslinker was introduced for high performance reversed-phase liquid chromatography (RPC). The co-polymerization was performed in situ in a stainless steel column of 4.6mm i.d. in the presence of a ternary porogen consisting of 1-dodecanol and cyclohexanol. This monolithic column (referred to as naphthyl methacrylate monolithic column or NMM column) showed high mechanical stability at relatively high mobile phase flow velocity indicating that the column has excellent hydrodynamic characteristics. To characterize the NMM column, different probe molecules including alkyl benzenes, and aniline, benzene, toluene and phenol derivatives were chromatographed on the column and the results in terms of k, selectivity and plate counts were compared to those obtained on an octadecyl silica (ODS) column in order to assess the presence of π–π and hydrophobic interactions on the NMM column under otherwise the same elution conditions. The NMM column offered additional π–π interactions with aromatic molecules in addition to hydrophobic interactions under RPC elution conditions. Run-to-run and column-to-column reproducibility of solute k values were evaluated, and percent relative standard deviation of <1% and ∼2–3.5%, respectively, were obtained. Six standard proteins were readily separated on the NMM column using shallow (30min at 1.0mL/min), steep (10min at 1.0mL/min) and ultra steep (1min at 3.0mL/min) linear gradient elution at increasing ACN concentration in the mobile phase using a 10cm×4.6mm i.d. column in case of shallow and steep linear gradients and a 3cm×4.6mm i.d. column for ultra steep linear gradient.

RP-HPLC METHOD DEVELOPMENT AND ITS VALIDATION FOR THE ESTIMATION OF FEXOFENADINE IN RAT PLASMA AND ITS APPLICATION FOR PHARMACOKINETIC STUDIES

A simple, sensitive and rapid reverse phase performance liquid chromatography (RP - HPLC) method was developed and validated for the determination of fexofenadine from small volumes of rat plasma. The Sample preparation was very simple and it involves protein precipitation method with ACN From the re sults of all the validation parameters and applicability of the assay . T he present method can be useful for pre - clinical pharmacokinetic studies of fexofenadine with desired precision and accuracy along with high - throughput. The developed analytical method is for fexofenadine in reverse phase chromatographic method utilizing C 18 column (250 mm × 4.6 mm , 5 µ). The peak was detected using a fluorescence detector set at Ex 220 nm and Em 280 nm . The mobile phase used was 1M triethylamine pH - 4.0: Acetonitrile in the ratio of 65:35. The retention time of fexofenadine was found to be 8.25 min utes . The total chromatogr aphic run time was 12.0 min utes . The flow rate of the mobile phase was 1.0 ml/min at room temperature . Method was found to be linear from 2 to 500 ng/ml for Fexofenadine (r 2 ≥ 0.9998) . The results indicate the bio - analytical method is linear, precise and a ccurate. The developed method was

Comparison of second-derivative spectrophotometry and HPLC for determination of amygdalin in wild apricot kernels

Amygdalin is a cyanogenic glucoside found in apricot kernels. To facilitate the exploitation and use of wild apricot resources, it is necessary to be able to rapidly and efficiently determine the amygdalin levels within their kernels. To do so, this paper compares two methods, one based on second-derivative UV spectrophotometry and the other on high- performance reversed-phase liquid chromatography (HPLC). The second-derivative spectrophotometry method effectively eliminates background noise and is straightforward to perform while retaining the accuracy of the HPLC method. It is likely to be a useful tool for the large-scale determination of amygdalin concentrations in wild apricot kernels and press cake.

Purification and Biochemical Characterization of Trypsin Inhibitor from Oyster

In this paper, the purification and biochemical characterization of the endogenous oyster (Crassostrea gigas) trypsin inhibitor were researched. A oyster trypsin inhibitor（OTI）has been purified by successive ammonium sulfate precipitation, gel filtration, affinity chromatography and high performance reversed-phase liquid chromatography. OTI has a molecular weight of approximately 5036 Da estimated by high performance size exclusive liquid chromatography. OTI was heat-, acid- and basic-stable competitive trypsin inhibitor. And OTI was double-head inhibitor with the inhibition constant (Ki) value of 1.644×10-2 mmol L-1. OTI was composed of nine kinds of amino acid, and rich in cysteine, alanine and glutamic acid. Furthermore, OTI can inhibit the proliferations of human lung adenocarcinoma A549 cell and human cervical cancer Hela cell

Separation of 9,10-anthraquinone derivatives: Evaluation of C18 stationary phases

Chromatographic study of natural products helps to determine their molecular composition and to identify their sources (biological, geographical, etc.). However, identifying anthraquinoids is still a challenge because this chemical family is composed of more than half a thousand molecules. In the present work, a series of C18 stationary phases were systematically studied to evaluate their separation potential for these compounds in high performance reversed phase liquid chromatography (RPLC). The stationary phases with various physicochemical properties were evaluated with respect to the separation of 30 standards. Selectivity of the studied stationary phases and symmetry of the obtained peaks were chosen as the evaluation criteria. Calculations of symmetry and separation capacity parameters have been proposed. These parameters are not dependent on the dimensions of the columns and were used for the classifications of phases. In conclusion, this study is intended to be a guide as to which stationary phases analysts can use for resolving separation of a complex mixture of anthraquinoids.

Simultaneous determination of 15 top-prescribed pharmaceuticals and their metabolites in influent wastewater by reversed-phase liquid chromatography coupled to tandem mass spectrometry

A fast and sensitive high performance reversed-phase liquid chromatography–tandem mass spectrometry method was developed and validated for the analysis of 15 prescription pharmaceuticals and four of their metabolites in influent wastewater. The selected pharmaceuticals belonged to various classes, such as angiotensin converting enzyme inhibitors, angiotensin receptor antagonists, calcium antagonists, β-blockers, antidepressants, analgetics, anticonvulsants, platelet antiaggregants, and cholesterol lowering agents. They were selected from the list of top-sold prescription pharmaceuticals in Belgium. The chromatographic separation was optimized in order to achieve suitable retention times, good resolution for analytes susceptible of mass spectrometric cross-talk and high sensitivity in one single run. All compounds eluted within 9 min on a Phenomenex Kinetex C18 column, based on a newly developed technology that allows a very narrow distribution of the core–shell particles, providing high separation efficiency. Sample preparation was executed with solid-phase extraction on Oasis MCX cartridges. The method was validated by assessing specificity, selectivity, lower limit of quantification (LLOQ), linearity, accuracy, precision, extraction recovery, and matrix effects following Food and Drug Administration guidelines. The method LLOQs ranged from 0.5 to 25 ng/L. Calibration curves and LLOQs were designed to provide a good analytical performance at concentrations expected in real influent wastewater samples for each target compound. Eight deuterated analogues were used as internal standards for quantification. The method was applied to influent wastewater samples collected from 17 different wastewater treatment plants throughout Belgium. Most of the analytes were measured in the samples at concentrations above LLOQ. Seven of the compounds were for the first time reported in influent wastewater. The newly developed analytical method is currently used to assess relationships between sales figures of pharmaceuticals and their corresponding concentrations in influent wastewater.

OPTIMIZATION OF HPLC METHOD FOR QUANTIFYING TETRACYCLINE RESIDUE IN HONEY

Tetracyclines are used for the treatment or prevention of American and European fouldbrood in bee colonies which are caused especially by two species of bacteria: Paenibacillus larvae larvae and Melissococcus pluton, respectively. In the present study a method for determination of residual tetracyclines in honey has been optimized and validated. It was used the reversed-phase high performance liquid chromatography (HPLC) with UV detection. Tetracyclines were extracted from honey with 0.01M sodium succinate buffer. For the clean up step, solid phase extraction using metal chelate resin was applied. Tetracyclines were eluted with buffer containing EDTA. Linearity, precision, recovery and sensitivity were satisfactory. The limit of detection was established at 5 µg/kg

Study on the level of environmental endocrine disruptors in serum of precocious puberty patients

To investigate the role of environmental endocrine disruptors (EEDs) in causing the precocious puberty. The blood samples were collected from 79 cases of precocious puberty patients and 42 cases of normal children. The concentrations of 4-nonylphenol (4-NP), 1, 1-dichloro-2, 2, bis (p-chlorophenyl) ethylene (p, p'-DDE) and di-2-ethylhexyl phthalate (DEHP) in blood serum samples were measured by using reversed-phase high performance liquid chromatography (HPLC). The volume of uterus and ovary, the bone density, and the content of estradiol (E(2)) in serum were determined at the same time. The contents of EEDs in blood serums of precocious puberty and the indices of the target organs were analyzed by using of correlation and regression. In normal control group, p, p'-DDE was detected in all the blood samples (14.93 - 40.39 ng/ml), but 4-NP and DEHP were detected in some samples (ND -6.77 ng/ml, ND -17.61 ng/ml). The levels of 4-NP, p, p'-DDE and DEHP in blood serum in precocious puberty group were notably increased than that in control group (P < 0.01). In precocious puberty group, there was a positive correlations between the 4-NP in volume of uterus and the volume of ovary and the density of bone (r = 0.394, 0.286, 0.237, P < 0.01); p, p'-DDE and volume of uterus also showed a the positive correlation (r = 0.306, P < 0.01). The influencing extent of 4-NP was 1.3 times to that of the p, p'-DDE. The normal children and the children with precocious puberty should be all contaminated by EEDs, and the later be exposured to more EEDs. There might exist a close relationship between EEDs and the precocious puberty, and EEDs should be an important factor in causing the disease. Different kinds of EEDs might have different influencing extents to the target organs.

MODIFIER INFLUENCE ON SELECTIVITY OF REVERSED-PHASE HPLC SYSTEMS

Correlation of partition constants of nine benzene derivatives in various gas – liquid systems, of which the solutions constitute binary mixtures of water and organic solvents methanol (MeOH), acetonitrile (ACN), tetrahydrofuran (THF), show high values of correlation factor. On the other hand, the correlation of retention, as log k (k – retention coefficient) of the same solutes in high performance reversed phase liquid chromatography (RPHPLC) systems with stationary phase of the C18 type and eluents of similar composition as liquids in the gas – liquid partition systems, reveal much smaller values of correlation factor. The comparison of these correlation data leads to the inference that interaction of the solute in the stationary phase, especially with sorbed modifier, is responsible for selectivity variation when the modifier is changed in the binary mobile phase of the RPHPLC system. This behavior is additionally confirmed by the retention correlation of phenolic acids for RPHPLC system with the modifiers investigated.

CHROMATOGRAPHIC DETECTION OF SESQUITERPENE LACTONES IN PARTHENIUM PLANTS FROM NORTHWEST ARGENTINA

Several chloroformic extracts containing some sesquiterpene lactones from autochtonous plants of Parthenium hysterophorus, collected in the province of Salta (Argentina), were separated and identified by high performance reversed-phase liquid chromatography with C18 column and propanol-water mobile phases, using gradient elution and concentrations of propanol larger than 10% (v/v). Lactones previously purified and characterized by H1 RMN, C13 RMN, IR, and mass spectrometry, were used as standards. The objective of the study was to check if the nature of the lactones was modified according to the altitude of the region where the plants were grown. Among the lactones, five were already described: coronopilin, hymenin, hysterin, parthenin, and tetraneurin A. p-Methoxybenzoic acid and two new ambrosanolides were also detected. Tetraneurin A and hymenin, on the one hand, and parthenin and coronopilin, on the other, were better resolved with a mobile phase of 6% propanol, run during 45 min.

Investigations on Radiolabeling Substance P Derivatives and Reversed-Phase Liquid Chromatographic Separation of the Products

In investigations of radiolabeling substance P1–11 (SP) with 125I isotope, its [Tyr8]-derivative was subjected to the lactoperoxidase (LPO) technique, whereas the native substance P undecapeptide was coupled with 125I-tyrosin-N-hydroxy-succinimidyl ester to the corresponding 125I-Bolton-Hunter SP derivative. For separation of the desired components from their by-products, labeled [Tyr8]-SP was pre-purified by ion-exchange chromatography (IEC) on carboxymethyl cellulose and subsequently investigated by gradient high performance reversed-phase liquid chromatography (gRP-HPLC) with 0.1 M triethyl-ammonium phosphate in acetonitrile (pH 2.5) on an octadecylsilyl silica gel (RP-18) stationary phase. When the total amount of labeled peptide affording a specific activity of approx. 2 × 106 Ci/M was checked by gRP-HPLC, the LPO technique yields about an equimolar mixture of 125I-[Tyr8]-(Met11)-SP and 125I-[Tyr8]-(Met11O)-SP. However, in contrast, substantial enrichment of 125I-[Tyr8]-(Met11)-SP was observed when more retained IEC fractions were analyzed. The 125I-Bolton-Hunter reagent was coupled in about 54 % yield to the SP undecapeptide at slightly alkaline conditions and separated from by-products only by gRP-HPLC yielding a specific activity of approx. 2 × 106 Ci/M. Both radiolabeled derivatives of SP are further characterized by their binding properties to a specific antibody raised in rabbits against synthetic SP1–11. When individual fractions of either radioiodinated [Tyr8]-SP or the corresponding 125I-Bolton-Hunter derivative were investigated with respect to their binding properties to the antibody, only those fractions coinciding with the maximum amount of radioactivity showed a satisfactory ratio of specific vs. non-specific antibody-binding, especially in the case of the 125I-Bolton-Hunter derivative. Therefore, the observed results are in good accordance with the existence of the desired target components.

Purification and partial amino acid sequences of a new presynaptic toxin and a cytotoxin from venom of pit veper Agkistrodon blomhoffii brevicaudus.

The technique of the reverse-phase performance liquid chromatography (RP-HPLC) was employed to separate and purify the toxic proteins from the venom of Agkistrodon blomhoffii brevicaudus collected in China. 3 toxic proteins marked as AgTx-1, AgTx-2 and AgTx-3 consisting of about 122 amino acid residues were screened. The toxicities (LD50) of the AgTx-1, AgTx-2 and AgTx-3 were 0.075, 0.51 and 6.6 mg per kg weight of mice respectively. Toxicological experiment in the chick biventer cervicis nerve-muscle preparation showed that the acetylcholine (Ach) sensitivity of the preparation was unchanged after the total failure of the indirect contraction caused by AgTx-1 and AgTx-2, suggesting that they were presynaptic blockers, namely beta-type of snake toxins. However, the amplitude of indirect contraction of the preparation was gradually reduced due to its incomplete relaxation caused by AgTx-3, indicating that it should belong to the category of cytotoxins. The partial amino acid sequences of 3 toxins have been established. It was found in ref. [1] that the sequences of the first 32 N-terminal amino acid residues of AgTx-1 and AgTx-2, as well as beta-agkistrodotoxin (beta-AgTx) reported previously were identical (the residue at the position 30 of beta-AgTx should be Trp). In view of the similarity in toxicities, and the amounts in the venom and other properties, it was concluded that AgTx-1 should be beta-AgTx and consequently was renamed beta 1-AgTx. AgTx-2 should be the isoform of beta 1-AgTx, and correspondingly named beta 2-AgTx.

Separation and quantitative determination of grape and wine procyanidins by high performance reversed phase liquid chromatography

AbstractA method is described for the separation and quantitative analysis of procyanidins in grape and wine. On a polyamide chromatographic column an initial elution with water at pH 7.0 enabled elimination of interfering phenolic acids. A second elution with acetonitrile/water (30:70 v) was performed to extract catechins. A third elution with acetone/water (75:25 v) was carried out to obtain procyanidins. The quantitative determination of procyanidins was done by high performance reversed phase liquid chromatography. The HPLC analysis was brought down to ∼55 min. The method is accurate and reproducible and has an acceptable extraction efficiency.

Estimating soil/sediment partition coefficients for organic compounds by high performance reverse phase liquid chromatography

High performance reverse phase liquid chromatography (HPRPLC) retention parameters were correlated to organic-carbon-normalized partition coefficient ( K oc ) values for sorption of various uncharged organic substances onto sediments and soils in an attempt to devise a model for predicting sorption behavior. The results reveal a relationship between the logarithm of HPRPLC retention time and log K oc that appears to provide a reasonable means for estimating partitioning properties from HPRPLC data. The technique was compared to methods which utilize octanol/water partition coefficients ( K ow ) and aqueous solubilities as surrogate predictors of sorption behavior and was found to yield superior correlations in all cases tested.

Determination of partition coefficients and aqueous solubilities by reverse phase chromatography—I: Theory and background

Water solubilities and octanol/water partition coefficients are widely used to predict partitioning and bioconcentration phenomena for hydrophobic organic pollutants in aqueous systems. This paper is the first in a two part series describing the application of high performance reverse phase liquid chromatography (HPRPLC) for indirect estimation of these two physicochemical parameters to facilitate environmental fate and transport predictions for organic compounds. In the first part, thermodynamic factors which control partitioning processes, water solubilities, and reverse phase retention behavior are discussed, and models for interlinking these three properties are summarized. The second part presents the results of aqueous solubility and octanol/water partition coefficient predictions for a number of organic contaminants from measurements of their HPRPLC behavior, and compares the modeling capabilities of some of the theoretical partitioning/solubility equations developed in the first paper.

The amino acid sequence of kinetensin, a novel peptide isolated from pepsin-treated human plasma: Homology with human serum albumin, neurotensin and angiotensin

A novel nonapeptide with neurotensin-like immunoreactivity was isolated from pepsin-treated human plasma by dialysis, ion-exchange chromatography and high performance reversed-phase liquid chromatography. The amino acid sequence was determined by automated gas-phase sequence analysis as Ile-Ala-Arg-Arg-His-Pro-Tyr-Phe-Leu. Sequence homology with human serum albumin and with the biologically active peptides neurotensin and angiotensin is demonstrated. The name proposed for this peptide is kinetensin.

Seminal prostaglandins in men with subnormal sperm motility and therapeutic treatment

The contents of prostaglandins in seminal plasma from a total of 73 men were evaluated. The subjects were grouped as follows: normospermic men, patients with impaired motility, patients with small untreated varicocelle and patients with impaired motility and Kallikrein therapy. Sperm density, morphology and motility were examined. High performance reversed phase liquid chromatography (HPLC) in combination with specific radioimmunoassays were used for the determination of PGE 2, PGI 2 and PGF 2α. There was a significant difference (p < 0, 025; F-test) between the PGI 2 concentrations in patients with impaired motility (5,6 ± 1,4 pg/mg protein) and normal men (8,8 ± 3,7 pg/mg protein). PGE 2 and PGF 2α were significantly different in patients with varicocele (p < 0,025, F-test). Wide ranges of prostaglandins occured in the Kallikrein-group with no significant differences. We conclude that: a) PGI 1 is an additional prostaglandin compound in seminal plasma. b)its measurement may not be useful as diagnostic parameter in subfertile men and c) Kallikrein has no influence on the prostaglandin content in seminal plasma and other seminal parameters as motility, motility index and sperm counts.