High-performance liquid chromatographic analysis of chlorophylls, pheophytins and carotenoids in virgin olive oils: chemometric approach to variety classification

This work evaluate the possibility to get from the quali-quantitative determination of the pigments contained in monovarietal olive oils (chlorophylls, pheophytins and carotenoids) and from the multivariate statistical analysis of these measures, parameters able to distinguish within the cultivars. The chemometric variables used have concurred to obtain preliminary interesting results. Liquid-phase distribution and solid-phase extraction/purification procedures has been compared: recoveries for both are resulted higher than 94% for all the pigment classes and the R.S.D. values were below 10%. HPLC analysis, allowing the simultaneous pigment determination, and fluorescence detection, allowing a better green pigments measure (detection limits from 5 to 80ppb), are revealed a fundamental solution.     

Supercritical fluid extraction of amino acids from birch (Betula pendula Roth) leaves and their liquid chromatographic determination with fluorimetric detection

A method for supercritical fluid extraction (SFE) of amino acids was adapted and optimal experimental conditions were selected for a matrix consisting of dry leaves. The matrix-dependent SFE method uses a mixture of MeOH-H(2)O-acetonitrile (10:10:1 v/v/v) as a modifier (0.5 mL in situ, 300 muL on-line) at 70 degrees C and 40 MPa and no HCl is needed as an entrainer. The amino acids were quantified using high-performance liquid chromatography with fluorimetric detection (HPLC/FLD) after gradient elution on Zorbax Eclipse AAA columns (4.6x150 mm, 3.5 mum) with aqueous Na(2)HPO(4 )buffer of pH 7.8 and ACN-MeOH-water as a mobile phase. In comparison with Soxhlet extraction, SFE gave higher recovery and selectivity, but it required longer extraction time (90 min) and it was more labor-intensive (clean-up step after the pre-concentration). Both methods should be used separately or in combination according to the matrix, number of samples, and levels of ballast compounds.     

Improved high performance liquid chromatographic method for determination of carotenoids in the microalga Chlorella pyrenoidosa

Microalgae have become an important commercial source of carotenoids and microalgae-derived functional foods are consumed by people worldwide. Therefore, an HPLC method was developed to discern the variety and content of carotenoids in the microalga Chlorella pyrenoidosa. The microalga sample was powdered, extracted, saponified and subjected to HPLC analysis. A mobile phase of methanol-acetonitrile-water (84:14:2, v/v/v) (A) and methylene chloride (100%) (B) with the following gradient elution was developed: 100% A and 0% B in the beginning, maintained for 14 min, decreased to 95% A in 25 min, 75% A in 30 min, 74% A in 35 min, 45% A in 50 min and returned to 100% A in 55 min. A total of 32 carotenoids were resolved within 49 min by using a C30 column with flow rate at 1 mL/min and detection at 450 nm. An internal standard beta-apo-8'-carotenal was used to quantify all the carotenoids. All-trans-lutein was present in exceptionally large amount (125034.4 microg/g), followed by cis isomers of lutein (27975.3 microg/g), all-trans-alpha-carotene (2465.8 microg/g), zeaxanthin (2170.3 microg/g), cis isomers of beta-carotene (2159.3 microg/g), all-trans-beta-carotene (2155.0 microg/g), cis isomers of alpha-carotene (1766.7 microg/g), beta-cryptoxanthin (334.9 microg/g), neoxanthin and its cis isomers (199.7 microg/g), neochrome (65.2 microg/g), auroxanthin (38.5 microg/g) and violaxanthin and its cis isomers (38.1 microg/g).     

Supercritical fluid extraction of macrocyclic lactone mycotoxins in maize flour samples for rapid amperometric screening and alternative liquid chromatographic method for confirmation

A rapid and simple method for the direct screening of macrocyclic lactone mycotoxins (zearalenone, ZON; alpha-zearalenol, alpha-ZOL; and beta-zearalenol, beta-ZOL) in maize flour samples is proposed. The sample screening method comprises supercritical fluid extraction (SFE) and clean-up on Florisil adsorption cartridge of the selected toxic compounds, followed by continuous flow electrochemical detection. Those samples for which the total concentration is close to or above the threshold limit established by legislation (0.200 mg kg(-1)) are subjected to preconcentration on C18 chromatographic material and liquid chromatographic separation for confirmation purposes. This confirmation method allows the determination of ZON, alpha-ZOL and beta-ZOL in the range between 30 and 300 microg kg(-1), with a average relative standard deviation lower than 5.2 in all cases.     

Improved liquid chromatographic method for determination of carotenoids in Taiwanese mango (Mangifera indica L.)

An HPLC method was developed to determine the various carotenoids in Taiwanese mango (Mangifera indica L.). Initially, the peel and seed of mangoes were removed, the pulps were cut into pieces, freeze-dried, ground into powder, extracted and subjected to HPLC analysis. A mobile phase of methanol-isopropanol (99:1, v/v) (A) and methylene chloride (100%) (B) with the following gradient elution was developed: 100% A and 0% B in the beginning, maintained for 15 min, decreased to 70% A in 45 min, maintained for 15 min and returned to 100% A in 65 min. A total of 25 carotenoids were resolved within 53 min by using a C-30 column with flow rate at 1 mL/min and detection at 450 nm. alpha-Carotene was used as an internal standard to quantify all the carotenoids. All-trans-beta-carotene was present in largest amount (29.34 microg/g), followed by cis isomers of beta-carotene (9.86 microg/g), violaxanthin and its cis isomers (6.40 microg/g), neochrome (5.03 microg/g), luteoxanthin (3.6 microg/g), neoxanthin and its cis isomers (1.88 microg/g), zeaxanthin (1.16 microg/g) and 9- or 9'-cis-lutein (0.78 microg/g).     

Supercritical fluid extraction: a novel method for the resolution of tetramisole

A new resolution method, based on the selective distribution of enantiomers between a chiral solid and an achiral supercritical fluid phase, is reported. The chiral solid phase is formed from the optically active dicarboxylic acid derivative, (2R,3R)-O,O′-dibenzoyltartaric acid, and the racemic base (tetramisole). A new method is also described for the enrichment of enantiomeric mixtures which have an enantiomeric ratio other than 1:1. This is based on the partial salt formation of the enantiomeric mixture with an achiral substance, which is then followed by supercritical fluid extraction of the free enantiomer. The extract has an enantiomeric composition which is different from the starting mixture. The method is applied to an enantiomeric mixture of tetramisole with hydrochloric acid.     

Supercritical fluid extraction and HPLC determination of relevant polyphenolic compounds in grape skin

The polyphenols determined are: (+)-catechin, (-)-epicatechin, rutin, quercetin and trans-resveratrol. Suitable conditions of supercritical fluid extraction were established using ethanol as a modifier of the polarity solvent (supercritical carbon dioxide). Final extraction conditions were: 20% v/v ethanol, 60degreesC, 250 bars and flow rate 2 mL/min. Static step time and dynamic step time were established using a selected grape skin sample. The extract was collected in water; the more polar polyphenols ((+)-catechin and (-)-epicatechin) remain in solution but rutin, quercetin and trans-resveratrol precipitate in this medium, thereby the solution of the extracted polyphenols was filtered. (+)-Catechin and (-)-epicatechin were determined in the liquid fraction, while the solid fraction, containing rutin, quercetin and trans-resveratrol, was solved with ethanol/H20 (40:60). HPLC determination was carried out at C18 stationary phase, with ethanol/water/acetic acid as mobile phases and UV-visible diode array detection. Due to the significant differences between the polarity of the polyphenols, two different mobile phases were used. An ethanol/water/acetic acid (5:93:2) mobile phase was used to determine (+)-catechin (280 nm) and (-)-epicatechin (280 nm). On the other hand, rutin (254 nm), quercetin (254 nm) and trans-resveratrol (306 nm) were resolved using ethanol/water/acetic acid (40:58:2) as mobile phase. Instrumental parameters were optimised and analytical parameters obtained. The analytical method was validated and applied to five different varieties of Vitis vinifera from the geographical area of Valencia.     

High-performance liquid chromatographic determination of ciprofloxacin in rat brain and cerebrospinal fluid

A novel high-performance liquid chromatographic method for the fluorometric determination of a newer quinolone, ciprofloxacin (CPFX), in rat brain and cerebrospinal fluid (CSF) was developed. CPFX in brain homogenate was extracted and injected onto a reversed-phase column without fluorescence derivatization. CSF was directly analyzed without the extraction procedure. Calibration curves were linear over the concentration ranges of 10 to 500 ng/g for brain and 5 to 500 ng/ml for CSF. The recoveries of CPFX added to brain were more than 97% with a coefficient of variation of less than 4%. The present method was sensitive and reliable enough to be utilized for detailed pharmacokinetic studies of CPFX in rat brain and CSF.     

Countercurrent chromatographic separation: a hydrodynamic approach developed for extraction columns

In countercurrent chromatography (CCC) both stationary and mobile liquids undergo intense mixing in the variable force field of a coil planet centrifuge and the separation process, like the separation in conventional solvent extraction column, is influenced by longitudinal mixing in the phases and mass transfer between them. This paper describes how the residence time distribution (or the elution profile) of a solute in CCC devices and the interpretation of experimental peaks, can be described by a recently developed cell model of longitudinal mixing. The model considers a CCC column as a cascade of perfectly mixed equal-size cells, the number of which is determined by the rates of longitudinal mixing in the stationary and mobile phases. Experiments were carried out to demonstrate the validation of the model and the possibility of predicting the partitioning behaviour of the solutes. The methods for estimating model parameters are discussed. Longitudinal mixing rates in stationary and mobile phases have been experimentally determined and experimental elution profiles are compared with simulated peaks. It is shown that using the cell model the peak shape for a solute with a given distribution constant can be predicted from experimental data on other solutes.     

Liposomal benznidazole: a high-performance liquid chromatographic determination for biodistribution studies

In this work, an isocratic high-performance liquid chromatographic method for quantitation of liposomal benznidazole (BNZ) in biological tissues is presented. The method comprises protein precipitation together with an efficient extraction of bulk or liposomal BNZ with acetonitrile-dimethylsulfoxide (1:1, v/v) at a 2:1 (extraction solvent-tissue matrix, v/v or /vw) ratio; the process is completed by a final precipitation with trichloroacetic acid. The resultant supernatants are assayed chromatographically using a Kromasil C18 (25- x 0.4-cm i.d., 100 A, 5- microm particle size), with an isocratic mobil phase consisting of acetonitrile-water (40:60, v/v), a flow rate of 0.9 mL/min, and detected at 324 nm. Bulk BNZ is used as a reference standard for the analysis of samples containing liposomal BNZ. The assay is linear over a concentration range of 0.75 (the lowest quantity of analyte determined with precision and accuracy of >or= 20%) to 25 microg/mL-g in all liquid and solid matrices. Within-day precision is better than 6.4% in plasma and 8.6% in liver, the same for the two assayed concentrations. Between-day precision is 5.4% and 12.3% in plasma and 9% and 6.9% in liver for the two assayed concentrations, respectively. The absolute recoveries range between 70% and 97%. Therefore, the method is accurate and precise to be employed for detection of minor quantities of liposomal BNZ in biological tissues.     

Supercritical fluid extraction of flumetralin in tobacco

Summary This work evaluates the level of Flumetralin residues in real tobacco samples. Conventional extraction methods were compared with SFE methods in both static and dynamic modes using CO₂ and CO₂ with modifier. Additionally, in the dynamic SFE mode, different collection approaches, such as collection at room temperature without solvent, collection at room temperature in the presence of solvent (hexane) and collection in an ice bath, without solvent, were studied. SFE showed itself to be a promising extraction technique for pesticide residues in tobacco samples.     

Supercritical fluid extraction and liquid chromatography-electrospray mass analysis of vinblastine from Catharanthus roseus

Supercritical fluid extraction using carbon dioxide modified with methanol, methanol-diethylamine, or methanol-triethylamine was used to extract vinblastine from the aerial portions of Catharanthus roseus. An HPLC-electrospray ionization (ESI)/MS analysis method was also developed to quantify the alkaloids in these extracts. Of the supercritical solvents evaluated, carbon dioxide-methanol-triethylamine (80 : 18 : 2) at 80 degrees C and 34.0 MPa greatly improved the supercritical fluid extraction (SFE) yield of vinblastine by as much as 76.4% over methanol extraction, while the other solvent conditions extracted the compound at yields less than 25% that of a methanol extraction. These results were confirmed by the robust HPLC-ESI/MS analytical method developed in this study.     

Supercritical fluid extraction for identification and determination of volatile metabolites from Angelica dahurica by GC-MS

The volatile components of Angelica dahurica were obtained by supercritical fluid extraction (SFE) method. These oils obtained were analyzed by GC-MS (identification and determination of metabolites). The compounds were identified according to their retention indices and mass spectra (electron impact (EI), 70 eV). The effects of different parameters, such as pressure, temperature, flow rate of CO(2), and the amount of modifier, on the SFE of A. dahurica oil were investigated. A total of 50 compounds of SFE extracts were identified. Phellopterin (PO), isoimperatorin (IO), imperatorin (IM), alloimperatorin (AM), byakangelicin, isooxypeucedanin, and pimpinellin were the major coumarin compounds identified in A. dahurica SFE extracts. The quantitations of PO, IO, IM, and AM were then accomplished. The calibration curves showed good linearity (R(2) >0.99) in the concentration ranges tested. The recoveries were higher than 85%, with RSDs less than 10%. The GC-MS method was successfully validated and applied to the quantitation of A. dahurica.     

Solid-phase extraction and direct high-performance liquid chromatographic determination of metoprolol enantiomers in plasma

A method is described by which underivatized metoprolol enantiomers in plasma can be quantitated by high-performance liquid chromatography with fluorescence detection. Samples are prepared for injection using a simple solid-phase extraction procedure which is essentially 100% efficient for all analytes. The metoprolol enantiomers are resolved using a cellulose tris(3,5-dimethylphenylcarbamate) chiral stationary phase and a hexane-ethanol-diethylamine mobile phase. Samples were tested for adaptability to autoinjection and found to be stable for at least 16 h after reconstitution in mobile phase. The automated method was determined to be precise and accurate for enantiomer concentrations as low as 10 ng base per ml and was successfully employed in a pharmacokinetic investigation.     

Solid phase extraction and high performance liquid chromatographic determination of doxophylline in plasma

A sensitive and selective high performance liquid chromatographic doxophylline assay with ultraviolet detection has been developed for plasma samples. The drug is isolated from biological samples with a reversed phase C18 disposable extraction column. Plasma standard curves are linear for concentrations of doxophylline from 0.03 to 10 mg/L. At the therapeutic range concentrations, the recoveries are better than 96.9%. The coefficients of variation for the procedure are 4.1% and 2.7% for the concentrations 0.03 mg/L and 10 mg/L, respectively. By this method, pharmacokinetic profiles are obtained for six adult volunteers.     

Solvent extraction of tricyclic amines from blood plasma and liquid chromatographic determination

The extraction of seven tricyclic antidepressant amines from human plasma at different pH values was investigated for dichloromethane, diethyl ether and hexane--1-pentanol (95:5). The amines were extracted as bases and back-extracted to sulphuric acid, 0.10 mol/l, prior to the separation by bonded-phase liquid chromatography. Ether and hexane--1-pentanol (95:5) were most suitable, tertiary amines being best extracted at pH 8, and secondary amines at pH 10. Using ether, both while 15 min ws sufficient for hexane--1-pentanol (95:5). UV detection allowed concentrations down to 10 ng in 1 ml of plasma to be determined. Three ammonium ions--octylammonium, dimethylammonium, and trimethylammonium--were added as modifiers to the mobile phase containing acetonitrile in phosphoric acid, 0.10 mol/l. In the concentration interval 0.010--0.030 mol/l all of the amine modifiers gave on Polygosil C8 peak asymmetry factors of sufficiently low magnitude, while on Li-Chrosorb RP-18 this was so only for di- and trimethylammonium in a concentration of 0.030 mol/l.