Liquid chromatographic enrichment ofN-acetylmethionine from casein hydrolysates for stable-isotope-ratio analysis of sulfur

Summary A chromatographic method has been developed for enrichment of methionine-bound sulfur in casein, for stable-isotope analysis. Casein is precipitated from milk samples and cleaved by acid hydrolysis in 6m hydrochloric acid at 95 °C. The amino acids released are converted into theirN-acetyl derivatives by addition of acetic acid anhydride. After lyophilization,N-acetylmethionine is separated from the accompanying components on octadecylsilica by use of a gradient prepared from 0.02m formic acid and methanol. The fractions containingN-acetylmethionine are pooled and freeze dried. Procedures for hydrolysis and derivatization were optimized to furnish the highest yields. The influence of the abundance ratio on the chromatographic separation is shown and discussed. From 1.0 g casein 16.5 mgN-acetylmethionine were isolated.     

High-performance liquid chromatographic analysis of ginsenosides inPanax ginseng extracts using glass-ODS column

Summary Octadecyl-porous glass was prepared and used as the packing for reversed-phase high-performance liquid chromatography. A mixture of ginsenosides, saponins of ginseng was analyzed with detection at 203 nm. Ginsenosides Rf, Rg₂, Rb₁, Rc, Rb₂, and Rd were separated with acetonitrile-water (27.5:72.5) as the mobile phase. A well-resoluted chromatogram of ginsenosides Ro, Rg₁ and Re was also obtained with acetonitrile-water (16.5:83.5). The whole separation was achieved in 12 min with a flowrate of 1 ml/min. Calibration curves of ginsenosides Rb₁, Rc, Rb₂, Rd, Rg₁ and Re were linear up to 5 μg. It can be concluded that the rapid and accurate analysis of ginsenosides is possible by the described method.     

Aqueous chromatographic system for the quantification of propofol in biological fluids using a temperature-responsive polymer modified stationary phase

A new method for the quantitative analysis of monkey serum propofol, which is widely used as an anaesthetic agent, was developed by utilizing a temperature-responsive polymer of N-isopropylacrylamide (NIPAAm) and butyl methacrylate (BMA) as the stationary phase of HPLC–fluorescence detection. This poly(NIPAAm-co-BMA) copolymer undergoes a reversible phase transition from a hydrophilic to a hydrophobic microstructure when triggered by change in the temperature. Also this chromatographic system is possible to separate the analytes by using only water as a mobile phase. A pretreatment of the serum (80 μL) was only solid-phase extraction, and the recovery rate of propofol and internal standard was more than 77%, respectively. This method covered the calibration range from 0.5 μg/mL to 10 μg/mL and allowed a reproducible quantification of the serum propofol in administrated monkey serum. The intra- and inter-assay relative standard deviations were less than 14.1%. In addition, there was good relationship of the quantification values between the developed method and the widely used reversed-phase HPLC method. Our developed method has proven to be useful for a simple analysis of propofol in clinical practice, because the avoidance of complicated mobile phase preparation was possible, and only temperature changing could regulate the retention time of the analyte. In addition, by using water instead of fossil fuel, it is the ideal analytical method according to green chemistry.     

Prediction of the chromatographic behaviour for a series of diuretic compounds

Summary The proportion of organic modifier and the pH of the acetonitrile-water mixtures used as mobile phases were optimized in order to separate a group of diuretic compounds covering a wide range of physyco-chemical properties. The Linear Solvation Energy Relationship (LSER) formalism based either on the multiparameter π^*, β and α scales or the single solvent polarity parameterE _T ^N , have been used to predict their chromatographic behaviour as a function of the percentage of acetonitrile in the eluent. Moreover, correlation established between retention and pH of the aqueous-organic mobile phases have been used to predict the chromatographic behaviour of the diuretic compounds studied as a function of the eluent pH. Linear correlation between a function of the eluent pH. Linear correlation between the chromatographic retention and theE _T ^N polarity parameter of mobile phases containing different percentages of organic modifier has been obtained Based on the knowledge of the acid-base dissociation constant the relation between retention and mobile phase pH has also been linearized. These relationship allowed an important reduction of the experimental retention data needed for developing a given separation and a great improvement in chromatographic optimization schemes.     

Degradation processes in the cellulose/<Emphasis Type="Italic">N</Emphasis>-methylmorpholine-<Emphasis Type="Italic">N</Emphasis>-oxide system studied by HPLC and ESR. Radical formation/recombination kinetics under UV photolysis at 77 K

Degradation processes of N-methylmorpholine-N-oxide monohydrate (NMMO), cellulose and cellulose/NMMO solutions were studied by high performance liquid chromatography (HPLC) and electron spin resonance (ESR) spectroscopy. Kinetics of radical accumulation processes under UV (λ = 248 nm) excimer laser flash photolysis was investigated by ESR at 77 K. Beside radical products of cellulose generated and stabilized at low temperature, radicals in NMMO and cellulose/NMMO solutions were studied for the first time in those systems and attributed to nitroxide type radicals ∼CH₂–NO^•–CH₂∼ and/or ∼CH₂–NO^•–CH₃∼ at the first and methyl ^•CH₃ and formyl ^•CHO radicals at the second step of the photo-induced reaction. Kinetic study of radicals revealed that formation and recombination rates of radical reaction depend on cellulose concentration in cellulose/NMMO solutions and additional ingredients, e.g., Fe(II) and propyl gallate. HPLC measurements showed that the concentrations of ring degradation products, e.g., aminoethanol and acetaldehyde, are determined by the composition of the cellulose/NMMO solution. Results based on HPLC are mainly maintained by ESR that supports the assumption concerning a radical initiated ring-opening of NMMO.     

High-performance liquid chromatographic determination of saponins fromHedera helix L. using a light-scattering detector

Summary A high-performance liquid chromatographic method for the simultaneous determination of bidesmosidic and monodesmosidic saponins from the leaves ofHedera helix L. using a light-scattering detector is proposed.A satisfactory separation of the bidesmosidic and monodesmosidic saponins is obtained in 25 min on a reversed phase RP18 using an acetonitrile-water mixture as eluant. Hederasaponin C and -hederin were found to be the major saponins of the leaves. The linearity of response, repeatability and reproducibility of the proposed method were tested. The detection limits for hederasaponin C and -hederin were 0,1 and 0.05 g/20 l respectively.     

Semi-preparative high-performance liquid chromatographic separation of ceralure and related isomers

Summary A semi-preparative high-performance liquid chromatographic procedure on 5-m silica was developed for the isolation of gram quantities of ethyltrans-5-iodo-trans-2-methylcyclohexane-1-carboxylate (B₁) and ethyltrans-4-iodo-trans-2-methylcyclohexane-1-carboxylate (B₂) for comparative evaluation as male Mediterranean fruit fly,Ceratitis capitata, attractants and for NMR studies. This procedure can also be used analytically to determine the content of B₁ (the attractive isomer) in ceralure, the ethyl 4- and 5-iodo-trans-2-methylcyclohexane-1-carboxylate mixture. 1,1-Dimethylethylcis-5-iodo-trans-2-methylcyclohexane-1-carboxylate (A) and 1,1-dimethylethylcis-4-iodo-trans-2-methylcyclohexane-1-carboxylate (C) were isolated and converted to their ethyl esters, thus supplying the fourtrans-isomers of ceralure.     

Development of an efficient method for the preparative isolation and purification of chlorophyll a from a marine dinoflagellate Amphidinium carterae by high-speed counter-current chromatography coupled with reversed-phase high-performance liquid chromatography

In our research into chlorophylls of marine dinoflagellates, chlorophyll a was separated rapidly from the hexane extract of Amphidinium carterae in three steps. The first step was silica gel column chromatography, where elution was performed with 0–50% ethyl acetate in n-hexane. The second was high-speed counter-current chromatography using a two-phase solvent system consisting of n-hexane–ethyl acetate–methanol–water (5:5:5:1, v/v), and the third step was preparative reversed-phase high-performance liquid chromatography using a solvent system of acetone–water (89:11, v/v). HPLC analysis showed that the purity of chlorophyll a from the second step was over 83%, and after the third it was over 99%. Thirty milligrams of chlorophyll a was isolated from a crude sample of 250 mg of chlorophylls, and its structure was identified by analyzing its MS, ¹H NMR and ¹³C NMR spectra.     

Ultra high performance liquid chromatography-time of flight mass spectrometry for analysis of avocado fruit metabolites: method evaluation and applicability to the analysis of ripening degrees

We have developed an analytical method using UHPLC-UV/ESI-TOF MS for the comprehensive profiling of the metabolites found in the methanolic extracts of 13 different varieties of avocado at two different ripening degrees. Both chromatographic and detection parameters were optimized in order to maximize the number of compounds detected and the sensitivity. After achieving the optimum conditions, we performed a complete analytical validation of the method with respect to its linearity, sensitivity, precision, accuracy and possible matrix effects. The LODs ranged from 1.64 to 730.54 ppb (in negative polarity) for benzoic acid and chrysin, respectively, whilst they were found within the range from 0.51 to 310.23 ppb in positive polarity. The RSDs for repeatability test did not exceed 7.01% and the accuracy ranged from 97.2% to 102.0%. Our method was then applied to the analysis of real avocado samples and advanced data processing and multivariate statistical analysis (PCA, PLS-DA) were carried out to discriminate/classify the examined avocado varieties. About 200 compounds belonging to various structural classes were tentatively identified; we are certain about the identity of around 60 compounds, 20 of which have been quantified in terms of their own commercially available standard.     

Qualitative and quantitative analysis of polycyclic polyprenylated acylphloroglucinols from Garcinia species using ultra performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight tandem mass spectrometry

Polycyclic polyprenylated acylphloroglucinols (PPAPs) are a group of natural products isolated from different Garcinia species with a wide range of important biological activities. In this study, an ultra performance liquid chromatography (UPLC) coupled to photodiode-array detection and quadrupole time-of-flight mass spectrometry (Q-TOF) method was developed to characterize 16 PPAPs in 10 Garcinia species. In source dissociation techniques based on cone voltage fragmentation were used to fragment the deprotonated molecules and multiple mass spectrometry (MS/MS) using ramping collision energy were used to further break down the resulting product ions. The resulting characteristic fragment ions were generated by cleavage of C1-C5 bond and C7-C8 bond through concerted pericyclic reaction, which is especially valuable for differentiating three types of PPAPs isomers. As such, two new PPAPs isomers present in minor amount in the extracts of Garcinia oblongifolia were tentatively characterized by comparing their tandem mass spectra to the known ones. In addition, an UPLC-Q-TOF-MS method was validated for the quantitative determination of PPAPs. The method exhibited limits of detection from 2.7 to 21.4 ng mL⁻¹ and intra-day and inter-day variations were less than 3.7% and the recovery was in the range of 89-107% with RSD less than 9.0%. This UPLC-Q-TOF-MS method has successfully been applied to quantify 16 PPAPs in 32 samples of 10 Garcinia species, which were found to be a rich source of PPAPs.     

Methods for the calculation of occupied volumes in glassy polymers: The lattice integration and the monte carlo methods

A new model for characterizing the free volume of a glassy polymer—gas systems is proposed. An improved method for the calculation of occupied volume per monomer unit was developed within the limits of this model. The model assumptions, error estimates and algorithm efficiencies are described. Using the example of polyvinyltrimethylsilane, it is shown that linear dependences of logarithms of the diffusion and the permeability coefficients on specific accessible volume for inert gases exist.     

Evaluation of gas chromatographic methods for the determination of <Emphasis Type="Italic">trans</Emphasis> fat

Consumption of trans fat has been associated with increased risk of coronary heart disease. For nutrition labeling purposes, the US Food and Drug Administration (FDA) defines trans fat as the sum of all the fatty acids with at least one nonconjugated double bond in the trans configuration. The FDA regulation states that label declarations of trans fat are not required for products that contain less than 0.5 g of trans fat per serving if no claims are made about fat, fatty acids or cholesterol. While attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FT-IR) provides reproducible measurements for samples containing more than 5% trans fat, methods based on gas chromatography (GC) are needed to measure lower trans fat levels. Trans fat quantitation by GC has recently been updated by considering more fatty acids, focusing more attention on fatty acids present in low amounts, and by using 100-m high-polarity capillary columns for optimal separation. The consistently high interlaboratory relative standard deviations (RSD, e.g., 21% at 1% trans fatty acids (TFA), 60% at 0.17% TFA), and intralaboratory RSD values (e.g., 10% at 1% TFA, 16% at 0.17% TFA) for trans fat at 1% or less of total fat reported in the collaborative study data for American Oil Chemists Society Official Method Ce 1h-05 suggest the need to carefully define the parameters associated with GC analysis of fatty acids.     

High-performance liquid chromatographic assay for simultaneous determination of tramadol and its active metabolite in human plasma. Application to pharmacokinetic studies

Summary A sensitive liquid chromatographic assay for the quantitative determination of the opioid analgesic tramadol and its active metabolite is described. Fluconazole was used as internal standard. The assay involved a singletert-butyl methyl ether extraction and LC analysis with fluorescence detection. Chromatography was at 30°C pumping an isocratic mobile phase of acetonitrile-water (19∶81, v/v) containing 0.06M NaH₂PO₄ and 0.05M triethylamine, adjusted to pH 7.90, at 1 mL min⁻¹ through a reversed-phase, 250×4 mm base-stable column. The limit of quantitation of tramadol and its active metabolite was 1 ng mL⁻¹, only 0.5 mL plasma sample was required for the determination. The calibration curve was linear from 1–1000 ng mL⁻¹. Intra and inter-day precision (C.V.) did not exceed 10%. Mean recoveries of 96.38% for tramadol and 96.62% forO-demethyltramadol with CVs of 0.43% and 1.46% were obtained. Applicability of the method was demonstrated by a pharmacokinetic study on normal volunteers who received 100 mg tramadol intravenously.     

LC with Novel Electrochemical Detection for Analysis of Monoamine Neurotransmitters in Rat Brain After Administration of (R)-Salsolinol and (R)-N-Methylsalsolinol

A novel electrochemical detector with an acetylcholine film has been developed for HPLC. The chemically modified electrode is an efficient electrocatalyst of (R)-salsolinol ((R)-Sal), (R)-N-methylsalsolinol ((R)-NMSal), and monoamine neurotransmitters, enabling highly sensitive detection. The electrode is also stable and long-lived. Combined with microdialysis sampling, HPLC with the novel detector enabled successful study of changes in the concentrations of monoamine neurotransmitters in rat brain after the injection of (R)-salsolinol and (R)-N-methylsalsolinol.     

High-throughput analytical strategy with combined planar and column liquid chromatography for improvement of the poppy (Papaver somniferum L.) with a high alkaloid content

Summary Poppy capsules with a high alkaloid content are of great importance to the pharmaceutical industry, because approximately 35 000 tons of straw (capsule with short stem), 350 tons of straw concentrate, and 1000 tons of opium are used annually for extraction of morphinane alkaloids. The combined use of four different liquid chromatographic methods is proposed for determination of alkaloid content. In the first, semi-quantitative, method screening is performed by multilayer overpressured-layer chromatography (MLOPLC) in which 142 samples are separated on silica as stationary phase within 5 s per sample. If the morphine content is >1.3% it is then measured quantitatively by NPHPTLC (normal-phase high-performance thin-layer chromatography) combined with densitometric evaluation. A second, quantitative, determination is always performed by means of a rapid RPHPLC (reversed-phase high-performance liquid chromatography) method in which the separation time is less than 4 min per sample. If the difference between the alkaloid content measured by use of the last two methods is >12% a confirmatory RPHPLC method with increased selectivity and analysis time (15 min) must be used. The high throughput strategy presented has been used for analysis of ca. 15 000 samples per year, per genotype. Systematic selection, cross-breeding, and this analytical strategy with combined planar and column liquid chromatographic methods resulted in the discovery of two new candidate cultivars with high morphine (ca. 2%) and thebaine (ca. 1.5%) content. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001.     

Use of a Graphical Method to Predict the Retention Times of Selected Flavonoids in HPLC from Thin-Layer Chromatographic Data

Similarities and differences between the retention characteristics of octadecylsilica wettable with water used in TLC and RP-18 used in HPLC have been elucidated by use of the linear relationships between log k and R_M. The stationary phases compared were investigated with the same mobile phases—binary mixtures of methanol and water, acetonitrile and water, and tetrahydrofuran and water. For these adsorbents of the same type but differing in specific surface area the correlation line was shifted by log (_systemI/_systemII). High values of the correlation coefficients obtained over the whole range of mobile phase organic modifier concentration examined indicated that the TLC systems could be used to predict HPLC conditions for flavonoid separation.     

Simultaneous Determination of Phytohormones in Plant Extracts using SPME and HPLC

Solid-phase microextraction followed by HPLC was used for the determination of indole-3-acetic acid (IAA), abscisic acid (ABA), indole-3-butyric acid (IBA) and 1-naphthylacetic acid (NAA) in plant samples. Parameters influencing performance, including pH, salinity, extraction time, fiber coating and temperature, were optimized. A Carbowax-coated fiber was chosen for determination due to much higher extraction efficiency compared to polyacrylate fibers. The dynamic ranges spanned over three orders of magnitude. The LOD/(LOQ) values of the target compounds in pure water were 0.149(0.497), 0.442(1.472), 0.121(0.403), 0.058(0.193) μg L⁻¹ for IAA, ABA, IBA and NAA respectively. The method was successfully applied to the analysis of xylem fluid from Musa basjoo stem obtaining recoveries of 98.85% (IAA), 94% (IBA) and 94.30% (NAA). The method was also successfully applied to the analysis of these four target compounds in the hyperaccumulating plant, Viola baoshanensis. The results matched quite well with ones obtained by solid phase extraction followed by HPLC. The method developed was superior when applied to liquid samples because matrix effects could be eliminated.     

Complexation equilibrium in a substrate- MgCl2(CaCl2)-Pd/C catalytic system and the influence of its position on asymmetric induction in hydrogenation ofN-acetyl-dehydrophenylalanyl-S-proline

The role of Ca and Mg salts as promoters of asymmetric induction in hydrogenation ofN-acetyl-dehydrophenylalanyl-S-proline in a substrate— MX₂—Pd/C catalytic system was studied. The data of potentiometric titration,¹H NMR spectroscopy, and the value of diastereomeric excess (de) of the resultingN-acetyl-R-phenylalanyl-S-proline indicate the complexation of the substrate with calcium and magnesium chlorides in an alcoholic solution. Ionization and equilibrium constants of the complex were determined. Excess salt shifts the equilibrium toward the complex formation, which increasesde up to 70 %.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 8, pp. 1964–1967, August, 1996.     

The use of1H—15N coupling constants for the investigation of tautomeric equilibria of azines

¹⁵N-¹H coupling constants can be measured over a large range by means of the Broadband-INEPT NMR-technique. These coupling constants are used to determine the positions of tautomeric equilibria of 2-(acetylmethyl)-azines with high accuracy.

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Die Anwendung von¹H—¹⁵N-Kopplungskonstanten bei Untersuchungen von Tautomerengleichgewichten von Azinen (Kurze Mitteilung)

Zusammenfassung Mittels der Breitband-INEPT NMR-Technik können¹⁵N-¹H-Kopplungskonstanten über einem weiten Bereich gemessen werden. Aus diesen Kopplungskonstanten kann die Lage der Tautomerengleichgewichte von 2-(Acylmethyl)-azinen mit großer Genauigkeit bestimmt werden.

     

HPLC-DAD and TLC-Densitometry for quantification of hypericin inHypericum perforatum L. Extracts

Summary Hypericum perforatum L. is a spontaneous perennial herbaceous plant, widely distributed in Europe, Asia, Northern Africa, and North America. The dried flowers or dried aerial parts are used to prepare the drug Hyperici Herba or St. John's Wort. Nowadays this drug is largely used as a natural antidepressant; hypericin and hypericin-like substances are considered the main active ingredients. In this work the hypericin and pseudohypericin content of hydroalcoholic extracts both of Hyperici Herba and ofHypericum perforatum L. dried flowers were measured by two techniques, TLC-densitometry with fluorescence detection and reversed-phase HPLC-DAD (diode-array detection). The quantitative data obtained by applying these techniques were compared and the identification of the main flavonoid constituents was performed by HPLC-DAD for characterization of the extracts. The quantitative data obtained by use of the two techniques were in good agreement and statistical analysis of the findings was indicative of the equivalence of the techniques.