Sequential Elution Research Articles

A simple method for differential isolation of freely dispersed and particle-associated peat microorganisms

Differential isolation of physiologically and ecologically diverse microbial groups facilitates evaluation of their activities in the environment. Here we describe a new method for differential isolation of freely dispersed and particle-associated peat microorganisms. The method is based on sequential elution of peat. Two types of natural peat, “young” sphagnum peat and “old” reed-based peat that have significantly different molecular composition as revealed by solid-state 13C NMR, were used for the method development. Various combinations of elution parameters (pH values, surfactant concentration, and sonication) were tested. Protein yield, CFU counts, 16S rDNA gene sequence, and T-RFLP analyses were used as criteria for evaluating the effectiveness of the elution protocols. The optimal method consisted of sequential elutions with (i) pH 6.2, 0.1 M potassium phosphate buffer, (ii) the same buffer amended with 0.01% of Tween 80, and (iii) treatment (ii) amended with sonication for 2 min. This method allowed differential isolation of freely dispersed and particle-associated microorganisms with (i) highest yield, (ii) highest diversity within the each microbial fraction, and (iii) best discrimination between the freely dispersed and particle-associated microbial communities. The method was applied for characterization of peat microbial communities and revealed different taxonomic composition and similar spatial organization of sphagnum and reed-sage peat microorganisms.

Simultaneous solid-phase extraction of acidic, neutral and basic pharmaceuticals from aqueous samples at ambient (neutral) pH and their determination by gas chromatography–mass spectrometry

Seven polymeric solid-phase extraction (SPE) sorbents were evaluated with regard to their ability to extract acidic, neutral and basic pharmaceuticals and estrogens simultaneously from water at neutral pH. Highest recoveries (70–100%) for the majority of the analytes were obtained with styrene–methacrylate and styrene– N-vinylpyrrolidone co-polymers. The latter one (Oasis HLB) was chosen for further refinement of an extraction method for the quantitative determination of acidic and neutral drugs in surface water samples at detection limits below 1 ng/l. A sequential elution protocol was applied for clean-up and separation of the extracted analytes into fractions suitable for further compound specific processing. The neutral analytes as well as the acidic compounds after derivatisation were quantified by GC–MS. Caffeine, ibuprofen, its metabolites and diclofenac were detected in river water samples in the 1–100 ng/l range.

Determination of isoxaflutole (balance) and its metabolites in water using solid phase extraction followed by high-performance liquid chromatography with ultraviolet or mass spectrometry.

Balance (isoxaflutole, IXF) belongs to a new family of herbicides referred to as isoxazoles. IXF has a very short soil half-life (<24 h), degrading to a biologically active diketonitrile (DKN) metabolite that is more polar and considerably more stable. Further degradation of the DKN metabolite produces a nonbiologically active benzoic acid (BA) metabolite. Analytical methods using solid phase extraction followed by high-performance liquid chromatography-UV (HPLC-UV) or high-performance liquid chromatography-mass spectrometry (HPLC-MS) were developed for the analysis of IXF and its metabolites in distilled deionized water and ground water samples. To successfully detect and quantify the BA metabolite by HPLC-UV from ground water samples, a sequential elution scheme was necessary. Using HPLC-UV, the mean recoveries from sequential elution of the parent and its two metabolites from fortified ground water samples ranged from 68.6 to 101.4%. For HPLC-MS, solid phase extraction of ground water samples was performed using a polystyrene divinylbenzene polymer resin. The mean HPLC-MS recoveries of the three compounds from ground water samples spiked at 0.05-2 microg/L ranged from 100.9 to 110.3%. The limits of quantitation for HPLC-UV are approximately 150 ng/L for IXF, 100 ng/L for DKN, and 250 ng/L for BA. The limit of quantitation by HPLC-MS is 50 ng/L for each compound. The methods developed in this work can be applied to determine the transport and fate of Balance in the environment.

Changes in proanthocyanidin chain length in winelike model solutions.

Reactions of seed and skin proanthocyanidins in the presence or absence of (-)-epicatechin were followed in winelike solutions over 53 days at 30 degrees C. Proanthocyanidins were separated from flavanol monomers by sequential elution from a Sep Pak cartridge, and changes in proanthocyanidin composition were monitored by thiolysis analysis of the proanthocyanidin fraction. In solutions containing no free (-)-epicatechin, trace amounts of monomers were released and important losses of proanthocyanidins were measured, but their average composition and mean degree of polymerization (mDP) were hardly modified. In the presence of (-)-epicatechin, the mDP value decreased and oligomeric proanthocyanidins accumulated throughout the incubation while losses of total units were dramatically reduced. Our data indicate that interflavanic bond cleavage of proanthocyanidins occurred under mild acidic conditions such as encountered in wine and that the resulting carbocation proceeded to unknown species. The latter reaction did not take place in the presence of (-)-epicatechin. Epicatechin added to the intermediate carbocation, thus being incorporated as the end unit of a shorter proanthocyanidin chain. The results of this study are discussed in relation to the loss of astringency reported during wine aging.

Pyrolysis‐gas chromatography/mass spectrometry of pitch fractions recovered from preparative size exclusion chromatography: structural differences with increasing molecular size

AbstractA sample of high‐temperature coal tar pitch has been fractionated by preparative scale size exclusion chromatography (SEC). Twenty‐four fractions corresponding to narrow molecular mass distributions were collected at sequential elution times of 3 minutes. The preparative scale column is thought to have filtered out most of the very large molecular mass material and the analysis focused on smaller mass components. The recovered fractions were characterised by analytical SEC. Pyrolysis‐gas chromatography/mass spectrometry (pyrolysis‐GC/MS) of the fractions was used to evaluate changes in main structural features with changing molecular size.As expected, analytical SEC of the fractions showed a systematic shift to smaller molecular sizes with increasing elution time. UV‐fluorescence spectra of the samples showed parallel structural shifts to shorter wavelengths and gains in intensity with increasing elution times. Pyrolysis‐GC/MS showed parent polycyclic aromatic components considered typical of pitch. The major fragments detected were pyrenes and fluoranthenes (m/z 202) and chrysenes (m/z 228). Benzopyrenes (m/z 252) and larger aromatics were not prominent amongst the pyrolysis products. Taken together, molecular structures were found to change significantly from fraction to fraction, but no systematic pattern emerged with changing elution time. Some components were identified as having formed during the analytical pyrolysis procedure itself. As aromatic products larger than dibenzopyrene (m/z 302) would not elute from the column, detected fragments are thought to have detached from larger pitch molecules.The pyrolysis of the largest species gave only alkanes and alkenes, with the aromatic fragments thought to be too large to elute through the GC column. The work provides information regarding the aromatic groups that are readily lost from larger molecules. Larger aromatic structures are thought to condense (to char) during the analytical pyrolysis step; the method is limited in not being able to provide information about their structural features. Copyright © 2002 John Wiley &amp; Sons, Ltd.

On-column trace enrichment by sequential frontal and elution electrochromatography: II. Enhancement of sensitivity by segmented capillaries with z-cell configuration—application to the detection of dilute samples of moderately polar and nonpolar pesticides

An on-column trace enrichment method for capillary electrochromatography of dilute samples is described. It involves the sequential use of frontal and elution electrochromatography on a segmented capillary column comprising of two contiguous segments each packed with a different sorbent. While the entering segment is for preconcentration by frontal electrochromatography the second segment is much longer and is meant for separation of the enriched analytes in the subsequent elution electrochromatography step. The preconcentration segment is usually packed with a sorbent that affords the highest affinity towards the solutes of interest while the separation segment is packed with a stationary phase that exhibits the highest selectivity and separation efficiency for the analytes. The detection is performed in the UV using a z-cell configuration for achieving an increased path length for detection. The effectiveness of this on-column trace enrichment is demonstrated on dilute samples of moderately polar solutes (e.g., carbamate insecticides) and nonpolar solutes (e.g., pyrethroid insecticides). Under optimal frontal and elution electrochromatography conditions, 817- and 1100-fold sensitivity increase are achieved for permethrin (a pyrethroid insecticide) and methiocarb (a carbamate insecticide), respectively, with a UV detector. The method is demonstrated with real water samples (e.g., tap and lake water samples) spiked with carbamate and pyrethroid insecticides. The limits of detection for the pesticides achieved in tap and lake waters reached 10 −8 to 10 −9 M.

Application of the sequential elution technique to determine Cd and Cu cellular location in Ulva lactuca Linnaeus.

The sequential elution technique, used to determine the cellular location of metals in nonvascular plants, was adapted to the seaweed Ulva lactuca Linnaeus for the heavy metals Cu and Cd. The optimum conditions for the extraction of extracellular Cu and Cd were determined using specimens previously loaded with these metals. The best extracellular metal extraction media and the number of extracellular metal extraction steps obtained were the following: Two steps (1 h + 40 min) in a filtered sea water-20 mM Ni solution for Cd and two steps in a filtered sea water-50 mM Co solution for Cu. To find these extraction conditions the extracellular metal extraction capacity of the different media used as well as the alteration provoked in plasmalemma permeability were compared. The concentrations of the incubation solutions of Cd and Cu that allowed reliable comparisons between the different extracellular metal extraction media used were previously determined.

SEPARATION OF CHICKEN EGG WHITE PROTEINS BY HIGH-SPEED COUNTERCURRENT CHROMATOGRAPHY

Chicken egg white proteins were fractionated by high-speed counter-current chromatography (HSCCC) using a cross-axis coil planet centrifuge (X-axis CPC). The separation was performed with an aqueous polymer two-phase system composed of 16% polyethylene glycol (PEG) 1000 and 12.5% potassium phosphate buffer at different pH of 6.8, 8.0, and 9.2. From 20 g of the crude egg white solution, the best separation was achieved with a polymer phase system at pH 8.0 by eluting the lower phase at 1.0 mL/min. After the sequential elution of ovotransferrin, ovalbumin, and lysozyme from the column, ovomucin still retained in the column was eluted in the reverse direction with upper PEG-rich phase. All four proteins in the CCC fractions were identified by SDS gel electrophoresis with Coomassie brilliant blue staining.

Identification of a defensin from the hemolymph of the American dog tick, Dermacentor variabilis

Hemolymph from partially fed virgin Dermacentor variabilis females was collected following Borrelia burgdorferi challenge and assayed for antimicrobial activity against Bacillus subtilis and B. burgdorferi. A small inducible cationic peptide was identified by SDS-PAGE in the hemolymph of these ticks as early as 1 h post challenge. Following purification by a three-step procedure involving sequential SepPak elution, reversed phase high performance liquid chromatography (RP-HPLC) and gel electrophoresis, the yield of the active peptide was approximately 0.1% of the total protein in the hemolymph plasma. The molecular weight, 4.2 kDa, was determined by MALDI-TOF mass spectrometry. N-terminal sequencing by the Edman degradation method gave a sequence for the first 30 amino acids as: G-F-G-C-P-L-N-Q-G-A-C-H-N-H-C-R-S-I-(R)-(R)-(R)-G-G-Y-C-S-Q-I-I-K. A computer search of databases showed that the peptide had 83% similarity to a defensin found in a scorpion. This is the first report of a defensin from a tick. The peptide was stable at least up to 70°C. Although the tick defensin alone was not immediately effective against B. burgdorferi, tick defensin plus lysozyme killed more than 65% of cultured B. burgdorferi within 1 h.

On-column trace enrichment by sequential frontal and elution electrochromatography. 1. Application to carbamate insecticides.

An on-column trace enrichment method for CEC of dilute samples is presented. The method involves on-line preconcentration by frontal electrochromatography under conditions of strong solute binding to the stationary phase followed by a step-gradient elution electrochromatography with a mobile phase of high eluting strength. This method is tested with dilute samples of carbamate insecticides using capillary columns of 100-microm i.d. packed with a 5-microm octadecyl silica (ODS) stationary phase. The effectiveness of on-line preconcentration (i.e., zone narrowing) depends on the retention factor, k', of the analyte in the injection solvent as well as in the eluting mobile phase (i.e., the organic solvent content), the applied voltage during sample introduction, and elution and length of the introduced sample plug. Under optimal frontal and elution electrochromatography conditions, a 500-fold sensitivity increase is achieved for carbofuran (a carbamate insecticide) with a UV detector. The method is demonstrated with deionized and tap water samples spiked with carbamate insecticides.

Determination of actinide elements at femtogram per gram levels in environmental samples by on-line solid phase extraction and sector-field-inductively coupled plasma-mass spectrometry

An on-line solid phase extraction method has been developed for the determination of 232Th, 237Np, 238U, 239Pu, 240Pu, 241Am and 243Am in biological certified reference material using a column containing TRU-Spec™ resin coupled with sector-field inductively coupled plasma-mass spectrometry. Absolute detection limits were 0.7, 0.85, 0.6, and 0.65 fg for 237Np, 239Pu, 241Am and 243Am, respectively. The 239Pu was determined in NIST Human Liver (963±297 fg g −1 compared with a certified value of 848±161 fg g −1) using a dry and wet ashing sample preparation method, and in a spiked cabbage reference material (394±54 fg g −1 compared to an indicative value of 467 fg g −1) using microwave digestion. Sequential separation of Pu and U was achieved by on-column reduction of Pu with titanium(III) chloride and elution in 4 M HCl to facilitate the determination of 239Pu in samples containing high levels of 238U, thereby eliminating the interference of 238U 1H + at m/ z 239. The sequential elution procedure was used to determine 239Pu in NIST human lung (814±55 fg g −1 compared with a certified range of 227–951 fg g −1) and NIST Rocky Flats Soil (2423±137 fg g −1 compared with a certified value of 3307±248 fg g −1).

DETERMINATION OF EIGHT LANTHANIDES IN APATITES BY ICP-AES, XRF, AND NAA

In the present work eight rare-earth elements (REEs), Hf, and Th were determined in eight apatite samples from San Luis, Argentina. The determinations were carried out by three methods: inductively coupled argon plasma atomic emission spectrometry (ICP-AES), neutron activation analysis (NAA), and X-ray fluorescence spectrometry (XRF). A new support for a small amount of sample was employed in the determination of La, Ce, and Nd by using XRF technique. The same procedure has proved reliable for the determination of major elements by using small amounts of apatite samples. For the ICP-AES determination, a group separation of the REEs from accompanying elements was required. This was achieved by ion- exchange chromatography and sequential acid elution. For the determination by NAA, a nondestructive instrumental technique (thermal neutron activation) was applied. Conditions of irradiation, decay, and measurements for the determination are described. Good correlation is obtained by these three methods.

Characterization of an in Vivo Hormonally Regulated Phosphodiesterase 3 (PDE3) Associated with a Liver Golgi-Endosomal Fraction

The biochemical properties of an in vivo hormonally regulated low Km cAMP phosphodiesterase (PDE) activity associated with a liver Golgi-endosomal (GE) fraction have been characterized. DEAE-Sephacel chromatography of a GE fraction solubilized by a lysosomal extract resulted in the sequential elution of three peaks of activity (numbered I, II, and III), while ion-exchange HPLC resolved five peaks of activity (numbered 1, 2, 3, 4, and 5). Based on the sensitivity of the eluted activity to cGMP and selected phosphodiesterase inhibitors, two phosphodiesterase isoforms were resolved: a cGMP-stimulated and EHNA-inhibited PDE2, eluted in DEAE-Sephacel peak I and HPLC peak 2 and a cGMP-, a cilostamide-, and ICI 118233-inhibited PDE3, eluted in DEAE-Sephacel peak III and HPLC peaks 3, 4, and 5. GE fractions isolated after acute treatments with insulin, tetraiodoglucagon, and growth hormone displayed an increase in phosphodiesterase activity relative to saline-injected controls, as did GE fractions from genetically obese and hyperinsulinemic rats relative to lean littermates. In all experimental rats, an increase in PDE3 activity associated with DEAE-Sephacel peak III and HPLC peaks 4 and 5 was observed relative to control animals. Furthermore, in genetically obese Zucker rats, an increase in the sensitivity of PDE activity to cilostamide and in the amount of PDE activity immunoprecipitated by an antibody to adipose tissue PDE3 was observed relative to lean littermates. These results extend earlier studies on isolated hepatocytes and show that liver PDE3 is the main if not sole PDE isoform activated by insulin, glucagon, and growth hormone in vivo.

THE SEPARATION OF Au(III) AND Pd(II) IN HYDROCHLORIC ACID SOLUTIONS BY STRONG ANION TYPE II EXCHANGE RESINS: THE EFFECT OF COUNTER ION CONCENTRATION AND TEMPERATURE

ABSTRACT The separation of gold and palladium ions in hydrochloric acid solutions by strong anion Type II exchange resins is studied in batch and column operations. Increases in the hydrochloric acid concentration and the temperature of the adsorption process have the effect of decreasing the adsorption capacity and the separation factor of both metals. A faster kinetics of the adsorption process of both ions results from temperature increase and this effect is higher for gold ions as a result of greater changes in its diffusivity. Although the adsorption capacity of palladium ions is lower at equilibrium conditions, the adsorption isotherms obtained suggest that they are more strongly retained by the polymers. The high capacities of these resins for both metals permits their use in the preconcentration of gold and palladium but their application in quantitative separation is restricted by the co-elution of up to% of the interfering ion during the desorption process. The best method for ion desorption is sequential elution with% ammonia for palladium recovery followed by 0·5M thiourea in HCI 1M for the recovery of gold.

A procedure for fractionation of sphingolipid classes by solid-phase extraction on aminopropyl cartridges

Solid-phase extraction (SPE) methods are easy, rapid, and reliable. Their growing popularity is in part due to their operational simplicity and cost reduction in solvents, and partly because they are easier to automate. Sphingolipids are implicated in various cellular events such as growth, differentiation, and apoptosis. However, their separation by small SPE cartridges has attracted limited attention. Here we describe an SPE procedure on aminopropyl cartridges that by sequential elution allows the separation of a lipid mixture into free ceramides, neutral glycosphingolipids, neutral phospholipids (sphingomyelin), and a fraction containing the acidic phospholipids and phosphorylated sphingoid bases, phosphoceramides and sulfatides. Individual components are obtained in high yield and purity. We applied the procedure to obtain data on separation of [(3)H]myristic acid-labeled sphingolipids from fish gills, and from human melanoma tumor tissue. Individual lipids in the SPE fractions were identified by chromatography on several high-performance thin-layer chromatography (HPTLC) systems. The chromatographic behavior of free sphingoid bases is also reported.

CHARACTERISATION OF METALFIX-CHELAMINE AND ITS APPLICATION IN PRECIOUS METAL ADSORPTION

ABSTRACT The physical and chemical characteristics of the chelating resin Chelamine, which contains a pentamine ligand, are investigated in order to consider its application in the separation of precious metals- Adsorption isotherm experiments for Pt, Pd and Au and kinetic experiments were carried out under batch procedures. The resin presents a high level of adsorption selectivity for Pt(IV), Au(III) and Pd(II) giving a capacity of 2.8 mmol/g, 3.1 mmol/g and 2.0 mmol/g respectively. Acidic solutions, complexing agents, lipophilic anions and ammonia were used for metal elution. NaClO4 solutions are the most effective eluting agents for Pt(IV) while thiourea is the best stripping agent for both Pd(II) and Au(III). Selective separation of the three metals can be achieved by sequential elution from the resin with NaClO4 solutions in different HCl concentrations and thiourea 0.5 M.

00/02394 Direct liquefaction of Wyodak coal using molybdenum sulfides as well as vanadium and tungsten-vanadium phosphomolybdates as catalyst precursors

In two separate coal conversion studies, one dealing with CO-water conversions of Illinois No. 6 coal, and the other with H2S addition to conventional tetralin conversions of the lower-rank Wyodak 2, data have been collected over a range of conversion levels. For both studies the degrees of conversion were increased by increasing the reducing power of the medium, while the conversion temperature was kept constant at 400 °C for all runs. The conversion levels for the CO-water runs ranged from 29 to 60%, while the tetralin-H2S runs yielded conversions from 44 to 67%. Sequential elution solvent chromatography and field ionization mass spectrometry were used for analyses of toluene-soluble fractions from the CO-water runs and THF-soluble fractions from the H2S-tetralin runs, respectively. The analyses showed that the fraction compositions remained constant with increasing degrees of conversion. This finding suggests that there may be some gross regularity in the structure of coal. Moreover, the results here are in contrast to work reported earlier, in which increased conversion levels, reached through greater conversion temperatures, yielded products with successively increased condensation and aromatization. It is thus suggested that severity be considered in terms of two components. Thermal severity reflects the temperature/time component of conversion chemistry, and derives from simple Arrhenius behaviour of competing reactions, while reduction severity reflects only on the reducing power of the system.

00/02396 Extraction of coal-tar pitches with toluene near the critical point: gasification and coal hydrogenated pitches

In two separate coal conversion studies, one dealing with CO-water conversions of Illinois No. 6 coal, and the other with H2S addition to conventional tetralin conversions of the lower-rank Wyodak 2, data have been collected over a range of conversion levels. For both studies the degrees of conversion were increased by increasing the reducing power of the medium, while the conversion temperature was kept constant at 400 °C for all runs. The conversion levels for the CO-water runs ranged from 29 to 60%, while the tetralin-H2S runs yielded conversions from 44 to 67%. Sequential elution solvent chromatography and field ionization mass spectrometry were used for analyses of toluene-soluble fractions from the CO-water runs and THF-soluble fractions from the H2S-tetralin runs, respectively. The analyses showed that the fraction compositions remained constant with increasing degrees of conversion. This finding suggests that there may be some gross regularity in the structure of coal. Moreover, the results here are in contrast to work reported earlier, in which increased conversion levels, reached through greater conversion temperatures, yielded products with successively increased condensation and aromatization. It is thus suggested that severity be considered in terms of two components. Thermal severity reflects the temperature/time component of conversion chemistry, and derives from simple Arrhenius behaviour of competing reactions, while reduction severity reflects only on the reducing power of the system.

In Situ extraction of rhizosphere organic compounds from contrasting plant communities

Currently there is no effective method for capture, identification and quantification of root exudates and rhizosphere secondary metabolites in situ. The purpose of the work reported was to assess if capsules containing non‐ionic carbonaceous resins could be used to non‐destructively sample and compare rhizosphere organic compounds associated with contrasting plant communities. Polyester capsules (Unibest, Inc., Bozeman, MT) containing non‐ionic carbonaceous resins, Ambersorb 563 or XAD‐7 (Rohm and Haas, Inc.), were placed within the rhizosphere of spotted knapweed (Centaurea maculosa), and the native grass, Idaho fescue (Festuca idahoensis), as well as a bare‐soil control in both greenhouse and field studies. At the end a 14‐day period, resins were removed and extracted with sequential elution by water, 50% methanol, and 100% methanol. The eluent fractions were then analyzed for total organic carbon (TOC), total hexose sugars as anthrone reactive carbon (ARC), and total phenols. Samples were then concentrated by rotary evaporating to dryness and analyzed on HPLC equipped with a C‐18 column and tunable ultra‐violet (UV) detector or a photodiode array (PDA) detector. Ambersorb 563 resin extracts from greenhouse and field trials consistently showed 2 times more soluble C and 3–7 times more total soluble sugars in the rhizosphere of knapweed compared to Idaho fescue during the 1996 and 1997 field seasons. This difference was not observed using the XAD‐7 resins during the rather wet field season of 1998. In these studies fescue was found to release higher levels of sugars than knapweed, but not significantly different than control soils. Compounds sorbed to the resins from the knapweed rhizosphere were more effectively eluted by methanol than water and demonstrated both the presence of carbohydrate groups and UV absorption. The XAD‐7 resins allow for sorption of phenolic compounds similar to that of Ambersorb 563, but allow for far greater desorption of these compounds. Non‐ionic resins may provide an effective means of capturing rhizosphere organic compounds in situ, but the low concentrations of sorbed compounds may limit their utility.

Determination of eight water- and fat-soluble vitamins in multi-vitamin pharmaceutical formulations by high-performance liquid chromatography

In the present work, a reversed-phase high-performance liquid chromatographic procedure has been developed for the determination of water-soluble vitamins (thiamine hydrochloride, pyridoxine hydrochloride, nicotinamide, riboflavin phosphoric ester and cyanocobalamine) and fat-soluble vitamins (retinol palmitate, cholecalciferol, α-tocopherol acetate) in multi-vitamin pharmaceutical formulations. The sample treatment proposed consists of a solid-phase extraction with C 18 AR cartridges that allow the separation of fat-soluble vitamins, which were retained on the sorbent, from water-soluble vitamins. Afterwards, the water-soluble vitamins were analysed by HPLC on a Nova-Pack C 18 (150×3.9 mm, 4 μm) analytical column, using CH 3OH–0.05 M CH 3COONH 4 as mobile phase The chromatographic analysis of the fat-soluble vitamins was carried out after their sequential elution with methanol and chloroform from C 18 sorbent, on the above column. The mobile phase employed was MeOH–CH 3CN (95:5, v/v) working at a flow-rate of 2 ml min −1 in isocratic mode. The solid-phase extraction for these vitamins had been previously optimised. The experimental variables studied were: application volume, elution solvents and cleaning solutions. The UV–Vis detection of vitamins was made at 270 nm for all the water-soluble vitamins (362 nm for B 12) and 285 nm for the water-soluble and fat-soluble vitamins present in real samples at different concentration levels. The accuracy of the method was tested obtaining an average recovery ranging between 78 and 116%.