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Worldwide, 25% of the population suffers from metabolic syndrome (MetS). The treatment of patients with MetS regularly includes drugs prescribed simultaneously to treat several disorders that manifest at the same time, such as hypercholesterolemia, arterial hypertension, and diabetes. To the authors&rsquo, best knowledge, there is no previous published analytical method for the simultaneous quantification of drugs used in the treatment of these diseases. In the present study, a rapid high-performance liquid chromatography with a diode-array detector HPLC-DAD methodology was developed for simultaneous quantification of carvedilol (CVD), telmisartan (TEL), bezafibrate (BZT), gliclazide (GZD), and glimepiride (GMP) in bulk and pharmaceutical form. The chromatographic separation of the five pharmaceuticals was achieved on a Hypersil GOLD C18 Selectivity (5 &micro, m, 150 &times, 4.60 mm2) using a mobile phase of acetonitrile (50%) and 0.02 M KH2PO4, pH 3 (50%) at a flow rate of 1 mL/min and at 25 &deg, C. The total separation time was 9 min. The analytical method was validated following the International Conference on Harmonization guidelines. A reproducible method was obtained with acceptable limits of detection (LOD) and quantification (LOQ) for CVD (0.012 and 0.035 &mu, g mL&minus, 1), TEL (0.103 and 0.313 &mu, 1), BZT (0.025 and 0.076 &mu, 1), GZD (0.039 and 0.117 &mu, 1), and GMP (0.064 and 0.127 &mu, 1). The validated method allowed the determination of these drugs in commercial pharmaceutical products both individually and simultaneously. The present method was found to be suitable for simultaneous quantification of the five drugs that are most commonly used in the simultaneous treatment of the metabolic syndrome.

peer-reviewed, The selection of the most promising anticancer compounds from the pool of the huge number of synthesized molecules is a quite complex task. There are many compounds characterization approaches which can suggest the best structural features of a molecule with the highest antiproliferative effect on the certain type of cancer cell lines. One of these approaches is the lipophilicity determination of compounds and the analysis of its correlation with the anticancer activity. Since the importance of the lipophilicity is underlined in many earlier studies, this study is focused on determination of lipophilicity of previously synthesized 17α-picolyl and 17(E)-picolinylidene androstane derivatives by using reversed-phase high performance liquid chromatography (RP-HPLC) as a very fast, effective and relatively cheap method. Determination of the chromatographic lipophilicity of the studied androstanes can be considered as the part of their physicochemical characterization, which is a very important step in their further selection as drug candidates. The present study does not neglect the in silico approach. The determined chromatographic lipophilicity was analyzed by quantitative structure-retention relationship (QSRR) approach in order to reveal which molecular characteristics contribute mostly to the typical behavior of the androstanes in the applied chromatographic system, and thus to their lipophilicity. Classical statistical approach and Sum of Ranking Differences method were used for selection of the best QSRR models which should be used in prediction of chromatographic lipophilicity of studied androstane derivatives., This study is financially supported by the research projects of the Ministry of Education, Science and Technological Development of the Republic of Serbia (No. 172012 and No. 172021) and the research project of the Provincial Secretariat for Science and Technological Development of Vojvodina (No. 114-451-347/2015-02).

Reversed-phase HPLC with mass spectrometric and diode array detection as well as some literature data were used to reveal the individual types of solutes in anthocyanin complexes of tulip flower petals that are responsible for tulip flower petals coloration of the samples available in the local flower market. It has been found that the main components of the complexes are 3-rutinosides and their 2”’ and 3”’ acylated with acetic acid derivatives of the three anthocyanidins - delphinidin, cyanidin and pelargonidin in the color dependent ratios, though trace quantities of 3-glucosides were found in some cases. For the anthocyanin structure confirmation a correlation analysis of solute retentions of cyanidin or pelargonidin derivatives vs that of delphinidin was proposed based upon equivalence of structures alteration in the solute pairs for each series. The specificity of solutes retention modes was revealed by relative retention analysis, the trend parameters reflected particularities of chromatographic behavior as well as that of electron spectra of the solutes. The difference of acylated anthocyanins retentions was proposed to disclose the conformation states of solutes in the sorbent interface.

Purpose: A simple and available reversed-phase high performance liquid chromatography (HPLC) method with UV detection has been developed and validated for mycophenolic acid (MPA) assay in human plasma. Methods: MPA was extracted from plasma with protein precipitation method by acetonitrile: percholeric acid: methanol (75:5:20 v/v/v). The drug separation was achieved using a C8 analytical column and a mobile phase of 0.1M triethylammonium phosphate (pH=5.4)-acetonitril (65:35, v/v), with a flow rate of 1.5 ml/min. The detection wavelength was 304 nm. Limit of detection (LOD) of the method was determined as the lowest MPA concentration producing a signal-to-noise (S/N) ratio of about 3. Limit of quantitation (LOQ) was determined as the lowest MPA concentration capable of being quantitated with enough accuracy and precision. Results: The method showed significant linear response-concentration relationship throughout the MPA concentration range of 0.2-10 µg/ml. A typical linear regression equation of the method was: y = 8.5523 x + 0.094, with x and y representing MPA concentration (in µg/ml) and peak height respectively, and the regression coefficient (r) of 0.9816. The average within-run and between-run variations of 7.81 and 4.78 percent. The average drug recovery from plasma was 95.24 percent throughout the linear concentration range. The limits of detection (LOD) and quantitation (LOQ) of the method were 0.05 and 0.2 µg/ml, respectively. The practical applicability of the method was proven throughout a bioequivalence study. Conclusion: The results showed the acceptable degree of linearity, sensitivity, precision, accuracy and recovery for the method. The method was used successfully for quantitation of MPA in plasma samples of healthy volunteers throughout a bioequivalence study.

International audience; Proanthocyanidins(PAs) are oligomeric and polymeric end products of the flavonoid biosynthetic pathway. They are present in the fruits, bark, leaves and seeds of many plants, where they provide protection against predation. At the same time they give flavor and astringency to beverages such as wine, fruit juices and teas, and are increasingly recognized as having a health promoting on human health. Seed extracts from five grape cultivars (Vitis vinifera) growing in El-Tarf (Algeria), were screened for their PAs composition and mDP (mean degree of polymerization). The study was realized by means of reversed-phase high-performance liquid chromatography coupled with photodiode array detector (RP-HPLC-DAD) analysis after thiolysis. The study revealed the presence of seven phenolic compounds belonging to the class of flavan-3-ol; Qualitative and quantitative differences among the cultivars were observed. The results confirm that grape seed of varieties studied are a potential source of PAs and can be used as easily accessible source of natural antioxidants.

The aim of this study was to develop and validate a fast and simple reversed-phase HPLC method for simultaneous determination of four cardiovascular agents—atorvastatin, simvastatin, telmisartan and irbesartan in bulk drugs and tablet oral dosage forms. The chromatographic separation was accomplished by using Symmetry C18 column (75 mm × 4.6 mm; 3.5 μ) with a mobile phase consisting of ammonium acetate buffer (10 mM; pH 4.0) and acetonitrile in a ratio 40:60 v/v. Flow rate was maintained at 1 mL/min up to 3.5 min, and then suddenly changed to 2 mL/min till the end of the run (7.5 min). The data was acquired using ultraviolet detector monitored at 220 nm. The method was validated for linearity, precision, accuracy and specificity. The developed method has shown excellent linearity (R2 > 0.999) over the concentration range of 1–16 µg/mL. The limits of detection (LODs) and limits of quantification (LOQs) were in the range of 0.189–0.190 and 0.603–0.630 µg/mL, respectively. Inter-day and intra-day accuracy and precision data were recorded in the acceptable limits. The new method has successfully been applied for quantification of all four drugs in their tablet dosage forms with percent recovery within 100 ± 2%.

Fat-soluble vitamins and antioxidants are of relevance in health and disease. Current methods to extract these compounds from biological fluids mainly need use of multi-steps and multi organic solvents. They are time-consuming and difficult to apply to treat simultaneously large sample number. We here describe a single-step, one solvent extraction of fat-soluble vitamins and antioxidants from biological fluids, and the chromatographic separation of all-trans-retinoic acid, 25-hydroxycholecalciferol, all-trans-retinol, astaxanthin, lutein, zeaxanthin, trans-β-apo-8'-carotenal, γ-tocopherol, β-cryptoxanthin, α-tocopherol, phylloquinone, lycopene, α-carotene, β-carotene and coenzyme Q10. Extraction is obtained by adding one volume of biological fluid to two acetonitrile volumes, vortexing for 60s and incubating for 60min at 37°C under agitation. HPLC separation occurs in 30min using Hypersil C18, 100×4.6mm, 5μm particle size column, gradient from 70% methanol+30% H2O to 100% acetonitrile, flow rate of 1.0ml/min and 37°C column temperature. Compounds are revealed using highly sensitive UV-VIS diode array detector. The HPLC method suitability was assessed in terms of sensitivity, reproducibility and recovery. Using the present extraction and chromatographic conditions we obtained values of the fat-soluble vitamins and antioxidants in serum from 50 healthy controls similar to those found in literature. Additionally, the profile of these compounds was also measured in seminal plasma from 20 healthy fertile donors. Results indicate that this simple, rapid and low cost sample processing is suitable to extract fat-soluble vitamins and antioxidants from biological fluids and can be applied in clinical and nutritional studies.

A series of the new urea derivatives with antinociceptive activity has been chromatographically evaluated using reversed phase materials: Zorbax Extend-C18, Cogent UDC Cholesterol with organic-aqueous eluent systems with two organic modifiers (methanol and acetonitrile). The chromatographic lipophilicity parameters: log kw, S and φ0 were determined basing on linear relationship between log k values and concentration of organic mobile phase modifier. The structure-retention studies revealed that the retention mechanism for all studied urea derivatives is uniform for the proposed chromatographic systems. However, a few exceptions were noticed. Derivatives containing nonpolar substituents in the imidazole ring acted as outliers for cholesterol column. In turn, the derivative containing ester polar substituent acted as an outlier in conventional reversed-phase system. Quantitative relationships based on a wide set of established computational molecular descriptors and experimental chromatographic data were also developed. Through a systematic study, by using the principal component analysis, fragmental method KowWIN appeared to be the most powerful software to produce reliable estimations of experimental retention parameters obtained on cholesterol column.

Reversed-phase HPLC with mass spectrometric and diode array detection as well as some literature data were used to reveal the individual types of solutes in anthocyanin complexes of tulip flower petals that are responsible for tulip flower petals coloration of the samples available in the local flower market. It has been found that the main components of the complexes are 3-rutinosides and their 2”’ and 3”’ acylated with acetic acid derivatives of the three anthocyanidins - delphinidin, cyanidin and pelargonidin in the color dependent ratios, though trace quantities of 3-glucosides were found in some cases. For the anthocyanin structure confirmation a correlation analysis of solute retentions of cyanidin or pelargonidin derivatives vs that of delphinidin was proposed based upon equivalence of structures alteration in the solute pairs for each series. The specificity of solutes retention modes was revealed by relative retention analysis, the trend parameters reflected particularities of chromatographic behavior as well as that of electron spectra of the solutes. The difference of acylated anthocyanins retentions was proposed to disclose the conformation states of solutes in the sorbent interface.

Procyanidins commonly known as condensed tannins are a type of polyphenol with wide abundance nat- urally. They are commonly known as potent anti-oxidants with powerful free radical scavenging activity as well as anti-tumor-promoting activity. Little is known about the enzymatic mechanisms/pathways involved in the microbial biotransformation of these polyphenolic molecules. The extracellular enzyme, dioxygenase produced by Aspergillus fumigatus was used as in vitro tools to study the degradation pathway of a model procyanidin dimer, namely procyanidin B2. The enzyme was purified to homogeneity by a two step process of anion-exchange chromatography coupled with FPLC followed by gel-filtration chromatog- raphy coupled with HPLC and the molecular mass estimated. In addition, the different biotransformed products resulted from the dioxygenase action on PB2 were purified using Reversed-Phase-High Perfor- mance Liquid Chromatography prior to their identification and characterization by structural elucidation using Electrospray Ionization-Mass Spectrometry. Subsequently, the mechanism of dioxygenase action on procyanidin dimer was defined. © 2010 Elsevier Ltd. All rights reserved.

Multidimensional chromatography coupled to mass spectrometry (LC(n)-MS) provides more separation power and an extended measured dynamic concentration range to analyse complex proteomics samples than one dimensional liquid chromatography coupled to mass spectrometry (1D-LC-MS). This review gives an overview of the most important aspects of LC(n)-MS with respect to optimizing peak capacity and evaluate orthogonality. We review recent developments in LC(n)-MS to analyse proteomics samples from the analyst point of view and give an overview over methods and future developments to process LC(n)-MS data for comprehensive differential protein expression profiling. Examples from our research, such as combining protein fractionation using high temperature reverse phase (RP) columns followed by analysis of the trypsin-digested fractions by RP LC-MS, serve to highlight possibilities and shortcomings of present-day approaches. Other LC(n)-MS systems that have been used to analyse highly complex shotgun proteomic samples, such as the combination of RP columns using low and high pH eluents or the combination of hydrophilic interaction liquid chromatography (HILIC) with RP-MS is discussed in detail.

A stability-indicating reversed-phase high-performance liquid chromatography (RP-HPLC) method has been developed which can separate and accurately quantitate paracetamol, dantrolene, cetirizine and pseudoephedrine. The method was successfully validated for the purpose of conducting stability studies of the four analytes in quality control (QC) laboratories. The stability-indicating capability of the method was demonstrated by adequate separation of these four analytes from all the degradant peaks. A gradient mobile phase system consisting of (A) 50 mmol L(-1) sodium dihydrogen phosphate, 5 mmol L(-1) heptane sulfonic acid sodium salt, pH 4.2 and (B) acetonitrile was used with Discovery reversed-phase HS C(18) analytical column (250 mm x 4.6 mm i.d., 5 microm particle size). Quantitation was achieved with UV detection at 214 nm, based on peak area. The proposed method was validated and successfully applied for the analysis of pharmaceutical formulations and laboratory-prepared mixtures containing the two multicomponent combinations.

A rapid extraction method involving microwave assisted extraction (MAE), followed by sample clean-up on a silica cartridge, reversed-phase high performance liquid chromatography (RP-HPLC) and spectrofluorimetric detection, was optimised for polycyclic aromatic hydrocarbon (PAH) determination in smoked meat. Compared to solvent extraction assisted by sonication, MAE, carried out with n-hexane on 2g of lyophilised sample at 115°C for 15min, allowed to obtain better extraction efficiencies. Limits of quantification (LOQ, s/n=10) lower than 0.2μg/kg wet weight were found for all PAHs, except for Fl (0.3μg/kg), P (0.6μg/kg) and IP (0.4μg/kg). The optimised procedure, that presented good analytical performances (with recoveries ranging from 77% to 103%, and precision within 10% for most of the PAHs), was applied to determine PAH content in different smoked meat products from the Italian market.

Auteur de correspondance: harry.gruppen@wur.nl; International audience; In order to fully explore the biofunctional potential of proanthocyanidins (PA), isolated and well-characterised PA dimers are of great importance. Current methods to obtain pure A- and B-type dimers are laborious, because they comprise multiple chromatographic steps, often yielding only one or two specific dimers. In the current study, an efficient isolation procedure is described, to isolate a large variety of A-type dimers from peanut skins and B-type dimers from grape seeds. Yields increased 20-400 times for A-type dimers and about 10 times for B-type dimers compared to other methods with a lesser number of chromatographic steps. Dinners isolated from peanut skins were identified as; epicatechin-(2-O-7, 4-8)-catechin (A1), epicatechin-(2-O-7, 4-8)-epicatechin (A2), epicatechin-(2-O-7, 4-6)-catechin, epicatechin-(2-O-7, 4-8)-entcatechin, isolated from peanut skins for the first time, and epicatechin-(4-6)-catechin (B7). Dimers from grape seeds were identified as; epicatechin-(4-8)-catechin (B1), epicatechin-(4-8)-epicatechin (132), catechin-(4-8)-catechin (B3) and catechin-(4-8)-epicatechin (B4)

Simple sample preparation procedures involving sonication and solid phase extraction (SPE), followed by reversed-phase high performance liquid chromatography (HPLC) and spectrofluorometric detection, were used to analyse polycyclic aromatic hydrocarbons (PAHs) in soil and olives collected in areas contaminated with creosote-treated railway ties. Very high PAH contents (with amounts ranging from 114.7 to 2157.2 and from 167.3 to 3121.8 μg kg− 1 dry weight for total light PAHs and total heavy PAHs, respectively) were found in soil sampled up to 1 m from the source of contamination. The PAH load decreased rapidly with the distance from the railway ties. High amounts of light PAHs, up to 6359.9 μg kg− 1, were also found in oil extracted from olives collected in a rural area where old railway ties were stored. No appreciable transfer of heavy PAHs and benzo[a]pyrene was observed in oil samples.

The development of highly potent chiral discrimination methods that solve the problems of the diastereomer method, in which it is impossible to discriminate the diastereomers having chiral centers separated by more than four bonds, is described. On the basis of the results obtained, a new hypothesis, Induced Chiral Fields that the achiral reversed phase can provide chiral fields depending on the structures of the eluents, is proposed to explain the significant results of separation of the diastereomers derived from newly developed chiral and fluorescent labeling reagents and optical isomers by reversed-phase HPLC, which was hitherto impossible.

The simple and rapid RP-HPLC method, for the simultaneous determination of lidocaine and cetrimonium bromide in the presence of pellet color corrigent, was developed. Separations were performed on a Beckman Ultrasphere ODS 4.6 mm x 15 cm, 5 microm particle column at 40 degrees C. The mobile phase consisted of water phase and acetonitrile (72:28 V/V), pH value of the mobile phase was adjusted to 2.0 with 85% ortophosphoric acid. Bisacodil was used as an internal standard. The flow rate was 1 ml/min and UV detection was performed at 208 nm. The proposed RP-HPLC method was validated and all the parameters for the validation of the method are given. According to the obtained results, the developed method was found to be suitable and accurate for the determination of these drugs in commercial formulations.

Separation and determination of n-alkylamines were investigated with anionic aldehydes to form water-soluble Schiff bases by using reversed-phase high-performance liquid chromatography (RP-HPLC). Two labeling reagents of salicylaldehyde-5-sulfonate (SAS) and 4-(4-sulfophenylazo)-1-hydroxy-2-naphthaldehyde (SPAHNA) were investigated. Sample solution containing the analytes and reagent solution were simultaneously injected and merged in the flow line, and water-soluble Schiff bases were formed in the reaction tube with its length of 5 m and at 70°C. The Schiff bases were resolved with water-methanol eluent on a reversed-phase column and photometrically detected. The entire analysis time including on-line derivatization, column separation and detection was accomplished within 25 min with SAS and 18 min with SPAHNA. The determination range for the n-alkylamines are 0∼50 μM with the limit of detections at 0.5∼1.0 μM for SAS and 0.08∼0.20 μM for SPAHNA., n-アルキルアミン類の分離定量法として，陰イオン性アルデヒドを誘導体化試薬として用いる吸光検出-逆相分配高速液体クロマトグラフィーを検討した．誘導体化試薬としては，サリチルアルデヒド-5-スルホン酸イオン（SAS）と4-（4-スルホフェニルアゾ）-1-ヒドロキシ-2-ナフトアルデヒド（SPAHNA）を用いた．n-アルキルアミンを含む試料溶液と試薬溶液はそれぞれの流路に同時に注入され，マージングゾーン法によりオンラインで混合され，5 m，70℃ の反応コイル中で水溶性シッフ塩基へと誘導体化された．生成した水溶性シッフ塩基は逆相カラムにより分離され，吸光検出された．オンライン誘導体化とカラム分離/検出を含む分析時間は，SASを用いた場合25分，SPAHNAを用いた場合は18分であった．アルキルアミン類に対する検量線は0～50 μMの範囲で良好な直線性が得られ，検出限界は，SASを用いた場合0.5～1.0 μM，SPAHNAを用いた場合は0.08～0.20 μMであった．

Four new triterpenoid estersaponins, tentatively designated as IIIA2 ( 1 ), IIIA3 ( 2 ), IIIB2 ( 3 ), and IIIC4 ( 4 ) have been isolated from fruits of Pittosporum tobira ait . and their structures elucidated by spectroscopic and chemical analyses. All the four compounds consist of an acylated pentacyclic triterpenoid aglycone which bears the same oligosaccharide portion. The crude saponin mixture (CIDI) was found to show significant in vitro and in vivo cytotoxicity in different human cancer cell lines.

Recently, a growing number of macromolecules such as peptides and proteins have been formulated into various microbicide formulations for the prevention of sexually transmitted infections. However, a fast and reliable high-throughput method for quantitating peptide/protein in polymer-based microbicide formulations is still lacking. As a result, we developed and validated a reversed-phase high-performance liquid chromatography method for the quantitation of gp120 fragment and LL-37 simultaneously in various microbicide gel formulations. This method was capable of detecting a limit of linearity (regression coefficient of 0.999) for gp120 fragment and LL-37 within a range of 0.625–80 and 1.25–80 µg mL−1, respectively. The lower limit of quantification for gp120 fragment and LL-37 was 1.14 and 0.31 µg mL−1, respectively. Method validation demonstrated acceptable intra- and inter-day RSD % (

The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.

解離不活性化学種だけを検出できるプレカラム誘導体化高速液体クロマトグラフィーを用いて, 閉殻希土類金属イオンの高感度・高選択的蛍光検出法を開発した. プレカラム誘導体化試薬として8-アミノ-2-[(2-アミノ-5-メチルフェノキシ)メチル]-6-メトキシキノリン-N, N, N', N'-四酢酸(Quin2)を用いた. 通常, ランタノイドイオン(Ln^)は解離活性化学種を形成するが, Ln^-Quin2錨体がプレカラム法で検出できたことは, これらの錯体が解離不活性化学種であることを示唆する. また, オンカラム錯形成モードHPLCにおける溶出挙動と異なり, 本システムにおける溶出順は中希土, 重希士, 軽希土類の順であった. また, 錯体の発光挙動は, La^,Lu^,Gd^及びY^だけが配位子由来の蛍光を有していた. これらの現象を利用し, La^, Lu^及びY^の分離検出を行い, それぞれの金属に対しほかのLn^の妨害なくppbレベルの検出限界を達成した.A sensitive and selective determination of closed-shell rare earth metal ions (La^, Lu^, and Y^) has been demonstrated using a pre-column chelating reagent {8-amino-2-[(2-amino-5-methylphenoxy)methyl]-6-methoxyquinoline-N, N, N', N'-tetraacetatic acid (Quin2)}, in reversed-phase HPLC. Although the resolution is still unsatisfactory in this HPLC system, rare- earth metal chelates with this octadentate aromatic polyaminocarboxylate can be detected. This result suggests that these chelates are unexpectedly inert for the dissociation process during elution. The order of elution is quite different from that of the conventional ion-exchange mode separation. Middle-lanthanide ion-Quin2 chelates were most rapidly eluted, followed by the heavy ones; the light ones were the most strongly retained. Since ligand-centered fluorescence was observed for only La^, Lu^, Y^, and Gd^-Quin2 chelates, and other rare earth metal ion chelates were sufficiently quenched through a paramagnetic deactivation process, these four metal ions were selectively detected by fluorimetric detection (λ_=335 nm, λ_ = 500 nm). The detection limits of La^, Lu^, and Y^ were 2.2, 4.0, and 1.9 ppb, respectively, on a 3σ basis. No interference from each 10-fold excess of other rare-earth metal ions was observed. The success of this HPLC system for rare- earth ions is derived from the combined properties of the Quin2 chelates in terms of inertness, chromatographic retention and fluorescence detection.