Reversed-phase Solid-phase Extraction Research Articles

Five year trends in the egg-thiamine status of Atlantic salmon from the St. Marys River, Michigan

Early mortality syndrome (EMS) is the term used to describe the mortality in early life stages of salmonids due to thiamine deficiency within the Great Lakes basin. Since 1985, Lake Superior State University (LSSU) has raised Atlantic salmon Salmo salar for stocking in the St. Marys River region near Sault Ste. Marie, Michigan. In 2000, the Atlantic salmon raised at LSSU experienced 99% mortality due to EMS related symptoms. In order to better understand and document the incidence of EMS in the St. Marys River population of Atlantic salmon, egg-thiamine content has been measured in this population since 2003. Egg-thiamine levels were measured using a rapid reversed-phase solid-phase extraction (RP–SPE) method of thiamine analysis. This paper presents the egg-thiamine data for this population of Atlantic salmon for the years 2003–2007. Over this five-year period, the egg-thiamine content of eggs from 2005 saw a significant increase. This one year shift may have larger ecological implications related to changes in the diet of this population of Atlantic salmon.

Roles for Stress-inducible Lambda Glutathione Transferases in Flavonoid Metabolism in Plants as Identified by Ligand Fishing

The glutathione transferases (GSTs) of plants are a superfamily of abundant enzymes whose roles in endogenous metabolism are largely unknown. For example, the lambda class of GSTs (GSTLs) have members that are selectively induced by chemical stress treatments and based on their enzyme chemistry are predicted to have roles in redox homeostasis. However, using conventional approaches these functions have yet to be determined. To address this, recombinant GSTLs from wheat and Arabidopsis were tagged with a Strep tag and after affinity-immobilization, incubated with extracts from Arabidopsis, tobacco, and wheat. Bound ligands were then recovered by solvent extraction and identified by mass spectrometry (MS). With the wheat enzyme TaGSTL1, the ligand profiles obtained with in vitro extracts from tobacco closely matched those observed after the protein had been expressed in planta, demonstrating that these associations were physiologically representative. The stress-inducible TaGSTL1 was found to selectively recognize flavonols (e.g. taxifolin; K(d) = 25 nM), with this binding being dependent upon S-glutathionylation of an active site cysteine. In the case of the wheat extracts, this selectivity in ligand recognitions lead to the detection of flavonols that had not been previously described in this cereal. Subsequent in vitro assays showed that the co-binding of flavonols, such as quercetin, to the thiolated TaGSTL1 represented an intermediate step in the reduction of the respective S-glutathionylated quinone derivatives to yield free flavonols. These results suggest a novel role for GSTLs in maintaining the flavonoid pool under stress conditions.

A rapid solid-phase extraction method for measurement of non-metabolised peripheral benzodiazepine receptor ligands, [18F]PBR102 and [18F]PBR111, in rat and primate plasma

To develop a rapid and reliable method for estimating non-metabolised PBR ligands fluoroethoxy ([(18)F]PBR102)- and fluoropropoxy ([(18)F]PBR111)-substituted 2-(6-chloro-2-phenyl)imidazo[1,2-a]pyridine-3-yl)-N,N-diethylacetamides in plasma. Rats and baboons were imaged with PET up to 2 h postinjection of [(18)F]PBR102 and [(18)F]PBR111 under baseline conditions, after pre-blocking or displacement with PK11195. Arterial plasma samples were directly analysed by reverse-phase solid-phase extraction (RP-SPE) and RP-HPLC and by normal-phase TLC. SPE cartridges were successively washed with acetonitrile/water mixtures. SPE eluant radioactivity was measured in a γ-counter to determine the parent compound fraction and then analysed by HPLC and TLC for validation. In SPE, hydrophilic and lipophilic radiolabelled metabolites were eluted in water and 20% acetonitrile/water. All non-metabolised [(18)F]PBR102 and [(18)F]PBR111 were in SPE acetonitrile fraction as confirmed by HPLC and TLC analysis. Unchanged (%) [(18)F]PBR102 and [(18)F]PBR111 from SPE analysis in rat and baboon plasma agreed with those from HPLC and TLC analysis. In rats and baboons, the fraction of unchanged tracer followed a bi-exponential decrease, with half-lives of 7 to 10 min for the fast component and >80 min for the slow component for both tracers. Direct plasma SPE analysis of [(18)F]PBR102 and [(18)F]PBR111 can reliably estimate parent compound fraction. SPE was superior to HPLC for samples with low activity; it allows rapid and accurate metabolite analysis of a large number of plasma samples for improved estimation of metabolite-corrected input function during quantitative PET imaging studies.

Quantitative determination of corticosteroids in bovine milk using mixed-mode polymeric strong cation exchange solid-phase extraction and liquid chromatography–tandem mass spectrometry

A new method was developed to identify and quantify corticosteroids (prednisolone, methylprednisone, flumetasone, dexamethasone, and methylprednisolone) in raw bovine milk by liquid chromatography–tandem mass spectrometry (LC–MS/MS) utilizing mixed-mode polymeric strong cation exchange and reversed-phase (MCX) solid-phase extraction (SPE) to reduce ion effects in a multimode ion (MMI) source. The main advantage of this method over other commonly used methods includes the use of a single SPE cartridge with a low volume for sample preparation and fast separation on the HPLC system with reduced ion suppression. This study is the first to report the determination of methylprednisone, a metabolite of methylprednisolone, in bovine milk. This method was validated in accordance with the European Union (EU) Commission Decision 2002/657/EC. The recoveries vary between 90% and 105%. The within-laboratory reproducibility (precision) is less than 30%. The decision limits and detection capabilities were calculated along with LODs, which ranged from 0.02 to 0.07 μg/kg. The method was further enhanced by its successful adaptation to other LC–MS/MS systems equipped with the newly developed ion source, Agilent Jet Stream (AJS). After optimization of the AJS ion source and MS parameters, even lower LOD values were achieved (0.001–0.006 μg/kg) for the corticosteroids. Analytical results obtained with the AJS were characterized by an enhanced area response and similar noise level comparable to those obtained with conventional orthogonal atmospheric ionization (API).

Kinetic electro membrane extraction under stagnant conditions—Fast isolation of drugs from untreated human plasma

Amitriptyline, citalopram, fluoxetine, and fluvoxamine were isolated by electro membrane extraction (EME) from 70 μl of untreated plasma (pH 7.4), through a supported liquid membrane (SLM) of 1-ethyl-2-nitrobenzene immobilized in the pores of a porous polypropylene hollow fiber, and into 30 μl of 10 mM HCOOH as acceptor solution inside the lumen of the hollow fiber. The driving force of the extraction was a 9 V potential sustained over the SLM with a common battery, with the positive electrode placed in the plasma sample and the negative electrode placed in the acceptor solution. Extractions were performed under totally stagnant conditions with a very simple device for 1 min (kinetic regime), and subsequently the acceptor solution was analyzed directly by liquid chromatography–mass spectrometry (LC–MS). Recoveries were 12, 13, 22, and 17% for fluoxetine, amitriptyline, citalopram, and fluvoxamine, respectively. Sample clean-up was comparable to reversed-phase solid-phase extraction (SPE), but EME required substantially less time than SPE. The time advantage of EME was further improved by parallel extraction of three samples (for 1 min) with the same 9 V battery. EME from plasma combined with LC–MS provided limits of quantification (S/N = 10) in the range 0.4–2.3 ng/ml, linearity in the range 1–1000 ng/ml with r 2-values of 0.998–0.999, and repeatability in the range 3.2–8.9% RSD in the mid-therapeutic window (100 ng/ml).

Elimination of LC–MS/MS Matrix Effect Due to Phospholipids Using Specific Solid-Phase Extraction Elution Conditions

The objective is to find elution conditions using different solid-phase extraction chemistries to reduce the amount of phospholipids in plasma extract, which is associated with matrix effect in LC-MS. Phospholipids' recovery was reduced by decreasing the eluents' methanol content applied to silica-based and polymer-based reversed-phase solid-phase extraction cartridges while 100% acetonitrile applied to the silica-based cartridges drastically minimized the phospholipids' elution. Silanol interactions are involved in the increased retention of phospholipids with silica-based reversed-phase cartridges when using high percentages of ACN in eluents. The bioanalytical usefulness of these findings was confirmed by successful extraction recovery of pharmaceutical compounds. Combinations of specific eluents and reversed-phase solid-phase extraction cartridges were found to prevent matrix effect due to phospholipids.

Simultaneous Quantification of Three Lipid Peroxidation-Derived Etheno Adducts in Human DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

Etheno DNA adducts are promutagenic DNA lesions derived from exogenous industrial chemicals, as well as endogenous sources including lipid peroxidation. Furthermore, levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues. In this study, we have developed a highly sensitive and specific stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) assay for simultaneous detection and accurate quantification of 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo), 3,N(4)-etheno-2'-deoxycytidine (epsilondCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in tissue DNA. Typically, [(13)C(1),(15)N(2)]epsilondAdo, [(15)N(3])epsilondCyd, and [(13)C(1),(15)N(2)]1,N(2)-epsilondGuo were added to calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The etheno adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by nanoLC-NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode. This nanoLC-NSI/MS/MS assay achieved attomole-level sensitivity with the detection limit of 0.73, 160, and 34 amol for the respective standard epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo injected on-column, while the quantification limit for the entire assay was 0.18, 4.0, and 3.4 fmol, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in human placental DNA were 28.2, 44.1, and 8.5 adducts in 10(8) normal nucleosides, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in 11 human leukocyte DNA samples were 16.2 +/- 5.2, 11.1 +/- 5.8, and 8.6 +/- 9.1 (mean +/- S.D.) in 10(8) normal nucleotides, respectively, starting from 30 mug of DNA or 1-1.5 mL of blood, and all the relative standard deviations were within 10%. An aliquot equivalent to 6 mug of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS/MS. Thus, this highly sensitive and specific nanoLC-NSI/MS/MS method is suitable for accurate quantification of the three lipid peroxidation-derived etheno DNA adducts as noninvasive biomarkers in clinical studies for cancer risk assessment and for evaluation of preventive agents.

Preliminary Characterization of Extracellular Allelochemicals of the Toxic Marine Dinoflagellate Alexandrium tamarense Using a Rhodomonas salina Bioassay

Members of the marine dinoflagellate genus Alexandrium are known to exude allelochemicals, unrelated to well-known neurotoxins (PSP-toxins, spirolides), with negative effects on other phytoplankton and marine grazers. Physico/chemical characterization of extracellular lytic compounds of A. tamarense, quantified by Rhodomonas salina bioassay, showed that the lytic activity, and hence presumably the compounds were stable over wide ranges of temperatures and pH and were refractory to bacterial degradation. Two distinct lytic fractions were collected by reversed-phase solid-phase extraction. The more hydrophilic fraction accounted for about 2% of the whole lytic activity of the A. tamarense culture supernatant, while the less hydrophilic one accounted for about 98% of activity. Although temporal stability of the compounds is high, substantial losses were evident during purification. Lytic activity was best removed from aqueous phase with chloroform-methanol (3:1). A “pseudo-loss” of lytic activity in undisturbed and low-concentrated samples and high activity of an emulsion between aqueous and n-hexane phase after liquid-liquid partition are strong evidence for the presence of amphipathic compounds. Lytic activity in the early fraction of gel permeation chromatography and lack of activity after 5 kD ultrafiltration indicate that the lytic agents form large aggregates or macromolecular complexes.

Improving the Determination of Nitrovin in Feeds by Reversed-Phase LC with SPE

A simple reversed-phase liquid chromatographic method with ultraviolet detector (378 nm) for the determination of nitrovin in feeds was improved and validated. The mobile phase was a mixture of acetonitrile and 0.1% formic acid solution (v/v) in the ratio of 50:50 (v/v), and the flow rate was set at 1.2 mL min−1. The extraction solution was a mixture of dimethyl formamide, acetonitrile and methanol (50:25:25, v/v), the sample was cleaned-up with reversed-phase solid phase extraction cartridge. The standard nitrovin was purified with crude nitrovin product by ethylene glycol monoethyl ether and identified by elemental analyzer. The limit of detection was 0.05 mg kg−1 and the limit of quatification was 0.2 mg kg−1 in feeds. The assay had satisfactory selectivity, recovery, linearity and precise repeatability and trueness.

A novel method to detect seven microcystins in hard clam and corbicula fluminea by liquid chromatography–tandem mass spectrometry

A simple and reliable method to detect seven microcystins in hard clam and corbicula fluminea, based on liquid chromatography with electrospray ionization and tandem mass spectrometry (LC–ESI-MS/MS), was developed and validated. The sample preparation procedure includes extraction of tissue by methanol, followed by cleanup on a reversed-phase solid phase extraction (SPE) cartridge. With the optimized method, recoveries were between 43.7% and 92.3% for hard clam, 54.3% and 93.8% for corbicula fluminea, the relative standard deviations (RSD) were less than or equal to 16.2% and 15.7% in hard clam and corbicula fluminea at spiking levels of 1 μg/kg, 2 μg/kg and 5 μg/kg for MC-RR, MC-YR, MC-LR, and MC-LY, and 2 μg/kg, 5 μg/kg and 10 μg/kg for MC-LA, MC-LW and MC-LF, respectively, the limits of quantitation (LOQ) of this method were ranged from 0.7 μg/kg to 2.0 μg/kg.

Polysaccharides as a Marker for Detection of Corn Sugar Syrup Addition in Honey

Honey is a natural product of high quality. However, because of its limited production and of its relatively high price, some beekeepers or unscrupulous traders do not hesitate to modify and falsify this natural product in order to try to increase its market value. Then, these involved falsification practices, for example intentional addition of cheap sugar syrup to honeys, are sometimes difficult to detect. An effective and simple analytical method is proposed in order to detect adulteration in honey by analysis of polysaccharide profiles. Samples were previously treated with reversed-phase solid phase extraction first to remove monosaccharides and small oligosaccharides and second to concentrate simultaneously traces of polysaccharides. A chromatographic separation using anion exchange stationary phase and pulse amperometric detection was further performed. Polysaccharide fingerprints (degree of polymerization from 11 to 17) were shown to be present in laboratory doped samples, and not detectable or present at very low concentrations in the authentic honey samples. Application to acacia, mountain polyfloral and polyfloral honeys enabled readily the detection of fraud resulting from deliberate addition of 1% of corn syrup.

Optimization and validation of a high-performance liquid chromatography method for the analysis of cardiac glycosides in Digitalis lanata

In this study, a simple and reliable HPLC method for the qualitative and quantitative analysis of cardiac glycosides in Digitalis lanata Ehrh. raw material was developed and applied to healthy and phytoplasma-infected plants. The target analytes cover a broad range of secondary metabolites, including primary, secondary and tertiary glycosides and the corresponding aglycones. The sample preparation was carried out by sonication of the plant material with 70% (v/v) aqueous methanol at room temperature, followed by reversed-phase solid-phase extraction purification from interfering pigments. The HPLC analyses were performed on a Symmetry C 18 column (75 mm × 4.6 mm I.D., 3.5 μm), with a gradient elution composed of water and acetonitrile, at a flow rate of 1.0 mL/min. The column temperature was set at 20 °C and the photodiode array detector monitored the eluent at 220 nm. The method was validated with respect to ICH guidelines and the validation parameters were found to be highly satisfactory. The application of the method to the analysis of D. lanata leaves indicated that air-drying was the optimum method for raw material processing when compared with freeze-drying. The analysis of healthy and phytoplasma-infected plants demonstrated that the secondary metabolite mainly affected by the pathogen presence was lanatoside C (153.2 μg/100 mg versus 76.1 μg/100 mg). Considering the importance of D. lanata plant material as source of cardiac glycosides, the developed method can be considered suitable for the phytochemical analysis and for the quality assurance of D. lanata used for pharmaceutical purpose.

Method development for the analysis of organophosphate and pyrethroid insecticides at low parts per trillion levels in water

In the current study, organophosphate and pyrethroid insecticides including diazinon, chlorpyrifos, bifenthrin, fenpropathrin, permethrin, λ-cyhalothrin, cyfluthrin, cypermethrin, esfenvalerate and deltamethrin were analyzed in laboratory and field-collected water samples. Water samples were extracted and analyzed by gas chromatography/electron capture detector (GC/ECD) and gas chromatography/nitrogen–phosphorous detector (GC/NPD). Comparison of results from liquid–liquid extraction and subsequent normal phase solid-phase extraction cleanup (LLE–NPSPE), and reversed phase solid-phase extraction (RPSPE) showed that LLE–NPSPE was the better choice to extract trace amounts of pesticides from water. Pesticide recoveries from four spiked water samples using LLE–NPSPE ranged from 63.2 to 148.8% at four spiking concentrations. Method detection limits were 0.72–1.69 ng/L using four different water sources. The stability of the target pesticides in lake water was investigated at 4 °C for 1 h, 1 d, 4 d, and 7 d under three conditions: (1) water samples only; (2) with 20 mL hexane used as a keeper solvent; and (3) with acidification to pH 2 with HCl. Results showed that water storage without treatment resulted in slow degradation of some pesticides with storage time, storage using water acidification led to significant degradation and loss of diazinon and chlorpyrifos, while water storage with hexane as a keeper solvent showed good stability for all of the target pesticides over the 7 d storage period.

Isoprenoid quantitation in human brain tissue: a validated HPLC-fluorescence detection method for endogenous farnesyl- (FPP) and geranylgeranylpyrophosphate (GGPP).

Farnesyl- and geranylgeranylpyrophosphate (FPP and GGPP) are isoprenoid intermediates in the mevalonate pathway. They play a crucial role in cell survival, growth and differentiation due to their attachment (isoprenylation) to small GTPases (Ras, Rho, etc.). Isoprenoid formation seems to be tightly regulated within the mevalonate pathway and its perturbation has been linked to certain diseases (e.g., cancer, Alzheimer's disease), but tissue levels are unknown. It is therefore of the utmost importance to quantify these isoprenoids in diseased tissue or in tissue after drug administration. The current work describes an isolation procedure utilizing a combination of Extrelut(R) liquid/liquid and reversed-phase solid-phase extraction (SPE) for homogenized human frontal cortex tissue. In addition, after a careful validation of an HPLC-fluorescence method, this assay allowed the determination of nanomolar concentrations of endogenous FPP and GGPP levels (4.5 and 10.6 ng/mg protein, respectively) in human brain tissue. The method is selective, precise (<15% RSD), accurate (<15% relative error) and sensitive over a linear range of 10-400 ng/mL for FPP and 50-1000 ng/mL for GGPP according to the current FDA criteria for bioanalytical method validation. Overall, this new method introduces the ability to simultaneously quantify FPP and GGPP in human brain tissue, and is potentially applicable to several other tissues and species.

Extraction of Aflatoxins B1 and G1 from Maize by Using Aqueous Sodium Dodecyl Sulfate

Aflatoxins are potent carcinogens produced by certain Aspergillus fungi. The aflatoxins were first discovered in the 1960s, and since then have been found to be distributed worldwide in a variety of commodities, foods, and feeds. Many of the early techniques for detecting aflatoxins involved extraction with halogenated solvents. With the increased availability and use of reversed-phase solid-phase extraction cartridges and the availability of immunoaffinity columns, aqueous mixtures of nonhalogenated solvents have been frequently used. To further reduce the need for solvents, we examined the effects of eliminating solvents during the extraction of maize, using aqueous mixtures of the detergent sodium dodecyl sulfate. After extraction and filtration, aflatoxins B1 (AFB1) and G1 (AFG1) were isolated by using commercially available immunoaffinity columns. The isolated AFB1 and AFG1 were derivatized with trifluoroacetic acid before separation by liquid chromatography with fluorescence detection. In spiked maize, the limits of detection were 0.5 and 1 ng/g for AFB1 and AFG1, respectively. Recoveries of AFB1 from maize spiked at 1-20 ng/g averaged 87.5% (range, 76.3-99.0%), with an average repeatability relative standard deviation (RSDr) of 4.0%. Recoveries of AFG1 from maize spiked at 2-20 ng/g averaged 80.4% (range, 70.3-85.8%), with an average RSDr of 3.5%. This is the first reported demonstration of an effective solvent-free extraction of aflatoxins from maize at ambient pressure, and this extraction procedure may serve to help reduce solvent consumption during aflatoxin analysis.

Low Molecular Weight Melanoidins in Coffee Brew

Analysis of low molecular weight (LMw) coffee brew melanoidins is challenging due to the presence of many non-melanoidin components that complicate analysis. This study focused on the isolation of LMw coffee brew melanoidins by separation of melanoidins from non-melanoidin components that are present in LMw coffee brew material. LMw coffee fractions differing in polarity were obtained by reversed-phase solid phase extraction and their melanoidin, sugar, nitrogen, caffeine, trigonelline, 5-caffeoylquinic acid, quinic acid, caffeic acid, and phenolic groups contents were determined. The sugar composition, the charge properties, and the absorbance at various wavelengths were investigated as well. The majority of the LMw melanoidins were found to have an apolar character, whereas most non-melanoidins have a polar character. The three isolated melanoidin-rich fractions represented 56% of the LMw coffee melanoidins and were free from non-melanoidin components. Spectroscopic analysis revealed that the melanoidins isolated showed similar features as high molecular weight coffee melanoidins. All three melanoidin fractions contained approximately 3% nitrogen, indicating the presence of incorporated amino acids or proteins. Surprisingly, glucose was the main sugar present in these melanoidins, and it was reasoned that sucrose is the most likely source for this glucose within the melanoidin structure. It was also found that LMw melanoidins exposed a negative charge, and this negative charge was inversely proportional to the apolar character of the melanoidins. Phenolic group levels as high as 47% were found, which could be explained by the incorporation of chlorogenic acids in these melanoidins.

Direct measurement of the glucuronide conjugate of 1-hydroxypyrene in human urine by using liquid chromatography with tandem mass spectrometry

To evaluate human exposure to polycyclic aromatic hydrocarbons (PAHs), we developed a rapid, simple and sensitive method for determining 1-hydroxypyrene-glucuronide (1-OHP-G) in human urine. To improve precision, a deuterated glucuronide was used as an internal standard. The method requires only 1 mL of urine. The urine was treated with a mixed-mode anion-exchange and reversed-phase solid-phase extraction cartridge (Oasis MAX). The analytes were analyzed with a C 18 reversed-phase column with a gradient elution, followed by tandem mass spectrometry with electrospray ionization in negative ion mode. The detection limit of 1-OHP-G (corresponding to a signal-to-noise ratio of 3) was 0.13 fmol/injection. Urinary concentrations of 1-OHP-G determined by this method were strongly correlated ( r 2 = 0.961) with concentrations of 1-hydroxypyrene by conventional HPLC with fluorescence detection.

Quantitative multiresidue method for about 100 veterinary drugs in different meat matrices by sub 2-μm particulate high-performance liquid chromatography coupled to time of flight mass spectrometry

A quantitative multiresidue method covering more than 100 veterinary drugs, belonging to different drug families, has been developed. The proposed approach uses an liquid–liquid–solid extraction technique (bi-polarity extraction) which is capable in recovering polar, medium polar and apolar compounds. A thorough generic reversed phase solid-phase extraction (SPE) clean-up removes interfering proteins and provides clean and stable extracts. Dedicated rinsing steps are proposed to reduce analyte adsorption on glass walls and on precipitating proteins. The resulting extract is analyzed by ultra-performance liquid chromatography (UPLC) coupled to time of flight mass spectrometry (TOF). The method was validated according to the Commission Decision 2002/657/EEC. Validation coved the relevant meat matrices (muscle, kidney and liver).

Rapid Isolation of Red Wine Polymeric Polyphenols by Solid-Phase Extraction

A rapid technique for the isolation of polymeric polyphenols from red wine has been developed and validated. A copolymer reversed-phase SPE cartridge was utilized in conjunction with predominantly organic eluents to provide three phenolic fractions from red wine without the need for sample pretreatment. The first fraction contained the bulk of the monomeric and oligomeric phenolic material, while the second and third fractions contained the polymeric polyphenolic compounds, as determined by HPLC analysis. The two polymeric polyphenolic fractions differed in their solubility and extent of pigmentation, and the differences appeared to be related to wine age. This method contrasted with other available fractionation techniques because the interfering, nonpolymeric material can be removed in a single wash fraction, while the polymeric material is separated into two distinct fractions based on their diverse physicochemical properties. It is anticipated that the rapid access to discrete polymeric fractions afforded by this method will be of benefit in furthering the understanding of red wine polymeric polyphenols.

Optimisation of programmable temperature vaporizer-based large volume injection for determination of pesticide residues in fruits and vegetables using gas chromatography–mass spectrometry

The applicability of programmable temperature vaporizer (PTV) solvent vent injection to the gas chromatographic (GC) determination of pesticide residues in fruits and vegetables was evaluated with the aim of miniaturizing the current multiresidue method. For that purpose 24 pesticides representing different chemical classes were initially chosen for optimisation of the large volume injection (LVI) parameters. Various parameters related to the optimum injector performance were tested for several types of packed and empty liners using both fast (at-once) and speed-controlled PTV solvent vent injection of standard solutions in ethyl acetate. In the next step, several packed and empty liners were evaluated for their suitability for pesticide multiresidue analysis. Parameters identified as optimal were then applied for PTV solvent vent injection of sample extracts prepared using the miniaturized multiresidue method to assess the long-term stability of the system. The combined use of large volume injection of 10 μl ethyl acetate extract into an empty multi-baffled or a CarboFrit packed liner using PTV injectors and GC–MS analysis enabled the detection and quantification of 124 pesticides in fruit and vegetable samples at the 0.01 mg/kg level using miniaturized reversed-phase solid-phase extraction (RP-SPE) of diluted acetone extract and clean-up on a small anion-exchange SPE column.