Liquid Chromatography With Diode Array Detection Research Articles

Production of enniatins A, A1, B, B1, B4, J1 by Fusarium tricinctum in solid corn culture: Structural analysis and effects on mitochondrial respiration

Enniatins (ENs) are secondary fungal metabolites with hexadepsipeptidic chemical structure and they possess a number of potent biological activities that can contaminate several kind of food and foodstuffs increasing the exposure risk for consumers. ENs are produced by several Fusariun strains including Fusarium subglutinans, Fusarium proliferatum and Fusarium tricinctum. Production of a mixture of ENs was performed by culturing F. tricinctum ITEM 9496 on white corn as substrate. The solid culture components were dried and subsequently extracted with water/methanol (50/50 v/v, 0.5% NaCl), homogenised, filtered, extracted by ethyl acetate and analysed by liquid chromatography with diode array detection (LC-DAD). The crude extract was first separated by low pressure liquid chromatography (LPLC) and then further purified by liquid chromatography (LC), resulting in six compounds with a purity higher than 95% as calculated by 1H NMR, and with a yield of 30–300mg per compound. The chemical structures of the ENs were determined by liquid chromatography coupled to mass spectrometry (LC–MS) and nuclear magnetic resonance (NMR). The biological activity of the resulting ENs was determined using a mitochondrial respiration test. We discovered that all the ENs studied induced an increase in the mitochondrial respiration resulting in uncoupling of the oxidative phosphorylation. This effect was most likely due to flux of K+ ions into the mitochondrial matrix. The order of potency of the ENs derivatives was: A1>B1>B>A>B4>J1. These results suggest a correlation between the chemical structures and bioactivity and confirm the severe risks for human associated with consumption of enniatins.

Development of methodology for determination of pesticides residue in water by SPE/HPLC/DAD

To boost crop yield, sugarcane growers are using increasing amounts of pesticides to combat insects and weeds. But residues of these compounds can pollute water resources, such as lakes, rivers and aquifers. The present paper reports the results of a study of water samples from the Feijão River, which is the source of drinking water for the city of São Carlos, São Paulo, Brazil. The samples were evaluated for the presence of four leading pesticides – ametryn, atrazine, diuron and fipronil – used on sugarcane, the dominant culture in the region. The samples were obtained from three points along the river: the headwaters, along the middle course of the river and just before the municipal water intake station. The pesticides were extracted from the water samples by solid-phase extraction (SPE) and then analyzed by liquid chromatography with diode array detection (LC-DAD). The analytical method was validated by traditional methods, obtaining recovery values between 90 and 95%, with precision deviations inferior to 2.56%, correlation coefficients above 0.99 and detection and quantification limits varying from 0.02 to 0.05 mg L−1 and 0.07 to 0.17 mg L−1, respectively. No presence of residues of the pesticides was detected in the samples, considering the detection limits of the method employed.

Development of a dispersive liquid–liquid microextraction method for the determination of fluoroquinolones in chicken liver by high performance liquid chromatography

A simple and cost effective sample pre-treatment method, dispersive liquid–liquid microextraction (DLLME), has been developed for the extraction of six fluoroquinolones (FQs) from chicken liver samples. Clean DLLME extracts were analyzed for fluoroquinolones using liquid chromatography with diode array detection (LC-DAD). Parameters such as type and volume of disperser solvent, type and volume of extraction solvent, concentration and composition of phosphoric acid in the disperser solvent and pH were optimized. Linearity in the concentration range of 30–500μgkg−1 was obtained with regression coefficients ranging from 0.9945 to 0.9974. Intra-day repeatability expressed as % RSD was between 4 and 7%. The recoveries determined in spiked blank chicken livers at three concentration levels (i.e. 50, 100 and 300μgkg−1) ranged from 83 to 102%. LODs were between 5 and 19μgkg−1 while LOQs ranged between 23 and 62μgkg−1. All of the eight chicken liver samples obtained from the local supermarkets were found to contain at least one type of fluoroquinolone with enrofloxacin being the most commonly detected. Only one sample had four fluoroquinolone antibiotics (ciprofloxacin, difloxacin, enrofloxacin, norfloxacin). Norfloxacin which is unlicensed for use in South Africa was also detected in three of the eight chicken liver samples analyzed. The concentration levels of all FQs antibiotics in eight samples ranged from 8.8 to 35.3μgkg−1, values which are lower than the South African stipulated maximum residue limits (MRL).

Principais técnicas de preparo de amostra para a determinação de resíduos de agrotóxicos em água por cromatografia líquida com detecção por arranjo de diodos e por espectrometria de massas

The determination of pesticide residues in water samples by Liquid Chromatography require sample preparation for extraction and enrichment of the analytes with the minimization of interferences to achieve adequate detection limits. The Solid Phase Extraction (SPE), Solid Phase Microextraction (SPME), Stir Bar Sorptive Extraction (SBSE) and Dispersive Liquid-Liquid Microextraction (DLLME) techniques have been widely used for extraction of pesticides in water. In this review, the principles of these sample preparation techniques associated with the analysis by Liquid Chromatography with Diode Array Detection (LC-DAD) or Mass Spectrometry (LC-MS) are described and an overview of several applications were presented and discussed.

Fast liquid chromatography-diode array detection assisted by chemometrics for quantification of seven ultraviolet filters in effluent wastewater

A fast chromatographic method is presented for simultaneous quantification of seven organic ultraviolet (UV) filters (benzophenone-3,4-methylbenzilidene camphor, octocrylene, 1-(4-tert-butylphenyl)-3-(4-methyoxyphenyl)1,3-propanedione), ethylhexyl methoxy cinnamate, ethylhexyl salicylate and homosalate) in effluent wastewater samples. The UV filters were pre-concentrated by Bond Elut-ENV cartridges and separated on an ODS column (15cm×0.46cm, 5μm) in less than 2.5min using a non-aqueous mobile phase of methanol–acetonitrile (50:50, v/v) with flow-rate of 1.5mLmin−1. Appropriate baseline correction through asymmetric least squares was applied to reduce the matrix of background signals in three way data. Then, second-order calibration based on multivariate curve resolution-alternating least squares (MCR-ALS) was implemented on the unfolded three-way data obtained from liquid chromatography with diode array detection (LC-DAD) through standard addition calibration method for handling co-eluted peaks, systematic and proportional errors. Recoveries ranging from 76% to 130% and %RSD values less than 11.2 for all UV filter shows the accuracy and precision of the proposed method in wastewater samples. In addition, statistical t-test as well as computed elliptical joint confidence region (EJCR) confirms the accuracy of the proposed method and indicates the absence of both constant and proportional errors in the predicted concentrations. This study demonstrates that coupling of the fast HPLC-DAD method with powerful algorithm of MCR-ALS can be considered as an efficient method for quantification of UV filters in highly contaminated samples of wastewaters where both time and cost per each analysis can be reduced significantly.

Exploiting second-order advantage using PARAFAC2 for fast HPLC-DAD quantification of mixture of aflatoxins in pistachio nuts

A new methodology using second-order calibration based on PARAllel FACtor analysis 2 (PARAFAC2) is developed for fast quantification of aflatoxins B1, B2, G1 and G2 in pistachio samples by liquid chromatography with diode array detection (LC-DAD). Solvent and solid phase extractions were used for sample preparation steps. In this work, a gradient elution programme was optimised to make co-elutions between analytes and also to shorten the analysis time. Prediction was made on synthetic and real validation samples with a mixture calibration design. Special circumstances such as overlapping of analytes, the lack of spectral selectivity and also the lack of trilinearity, have been compensated with “mathematic separation” through PARAFAC2 modelling. The results showed an acceptable performance of PARAFAC2 for resolution and quantification of all analytes in a single array exploiting the second-order advantage. With the proposed method, the chromatographic run time and also the time for data analysis were reduced significantly.

Science based calibration for the extraction of ‘analyte-specific’ HPLC-DAD chromatograms in environmental analysis

Multivariate science based calibration (SBC) has been applied to the resolution of overlapped peaks in liquid chromatography with diode array detection (LC-DAD). Complex river water samples spiked with 11 pharmaceutical substances resulted in poorly resolved chromatograms containing additional peaks from interfering matrix compounds and a change in the background absorbance due to the mobile phase gradient. Applying the present multivariate approach it was possible to resolve all 11 analytes from overlapping peaks, obtaining linear calibration lines (R2>0.96). Recovery percentages on spiked samples ranged between 74.6 and 113.5%, which are quite satisfactory taking into account the low concentration ranges considered to 1–7μgL−1.

New Reagent for Trace Determination of Protein-Bound Metabolites of Nitrofurans in Shrimp Using Liquid Chromatography with Diode Array Detector

The synthesis of derivatives of metabolites from furazolidone, furaltadone, nitrofurazone, and nitrofurantoin using a new derivatizing reagent, 2-naphthaldehyde (NTA), is described. The reaction product was used in liquid chromatography with diode array detector (LC-DAD) for determination of protein-bound metabolites of nitrofurans in shrimp followed by two steps of liquid-liquid extraction. Derivatives of nitrofuran metabolites are well separated from NTA remaining in the extract upon separation on a ChromSpher 5 Pesticide (250 x 4.6 mm, 5 microm) column at 40 degrees C with acetonitrile/5 mM ammonium acetate adjusted to pH 7.5 gradient as the mobile phase and DAD detection at 308 nm except for naphthyl derivative of 1-aminohydantoin at 310 nm. The high absorptivity of these derivatives makes simultaneous screening of these metabolites in shrimp at 1 microg/kg possible for the first time using LC-DAD. The method was validated using blank shrimp fortified with all four metabolites at 1, 1.5, and 2 microg/kg. Recoveries were >86% with relative standard deviations of <14% for all four metabolites. Comparison between LC-DAD and APCI-MS/MS shows very good agreement for shrimp samples.

Solving matrix-effects exploiting the second order advantage in the resolution and determination of eight tetracycline antibiotics in effluent wastewater by modelling liquid chromatography data with multivariate curve resolution-alternating least squares and unfolded-partial least squares followed by residual bilinearization algorithms: I. Effect of signal pre-treatment

The effect of piecewise direct standardization (PDS) and baseline correction approaches was evaluated in the performance of multivariate curve resolution (MCR-ALS) algorithm for the resolution of three-way data sets from liquid chromatography with diode-array detection (LC-DAD). First, eight tetracyclines (tetracycline, oxytetracycline, chlorotetracycline, demeclocycline, methacycline, doxycycline, meclocycline and minocycline) were isolated from 250 mL effluent wastewater samples by solid-phase extraction (SPE) with Oasis MAX 500 mg/6 mL cartridges and then separated on an Aquasil C 18 150 mm × 4.6 mm (5 μm particle size) column by LC and detected by DAD. Previous experiments, carried out with Milli-Q water samples, showed a considerable loss of the most polar analytes (minocycline, oxitetracycline and tetracycline) due to breakthrough. PDS was applied to overcome this important drawback. Conversion of chromatograms obtained from standards prepared in solvent was performed obtaining a high correlation with those corresponding to the real situation ( r 2 = 0.98). Although the enrichment and clean-up steps were carefully optimized, the sample matrix caused a large baseline drift, and also additive interferences were present at the retention times of the analytes. These problems were solved with the baseline correction method proposed by Eilers. MCR-ALS was applied to the corrected and uncorrected three-way data sets to obtain spectral and chromatographic profiles of each tetracycline, as well as those corresponding to the co-eluting interferences. The complexity of the calibration model built from uncorrected data sets was higher, as expected, and the quality of the spectral and chromatographic profiles was worse.

Quantitative metabolic profiles of tomato flesh and seeds during fruit development: complementary analysis with ANN and PCA

Tomato, an essential crop in terms of economic importance and nutritional quality, is also used as a model species for all fleshy fruits and for genomics of Solanaceae. Tomato fruit quality at harvest is a direct function of its metabolite content, which in turn is a result of many physiological changes during fruit development. The aim of the work reported here was to develop a global approach to characterize changes in metabolic profiles in two interdependent tissues from the same tomato fruits. Absolute quantification data of compounds in flesh and seeds from 8 days to 45 days post anthesis (DPA) were obtained through untargeted (proton nuclear magnetic resonance, 1H-NMR) and targeted metabolic profiling (liquid chromatography with diode array detection (LC-DAD) or gas chromatography with flame ionization detection (GC-FID)). These data were analyzed with chemometric approaches. Kohonen self organizing maps (SOM) analysis of these data allowed us to combine multivariate (distribution of samples on Kohonen SOMs) and univariate information (component plane representation of metabolites) in a single analysis. This strategy confirmed published data and brought new insights on tomato flesh and seed composition, thus demonstrating its potential in metabolomics. The compositional changes were related to physiological processes occurring in each tissue. They pointed to (i) some parallel changes at early stages in relation to cell division and transitory storage of carbon, (ii) metabolites participating in the fleshy trait and (iii) metabolites involved in the specific developmental patterns of the seeds.

Determination of phenolic acids in strawberry samples by means of fast liquid chromatography and multivariate curve resolution methods

Use of Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) is evaluated in the analysis of nine phenolic acids, both in standards mixture samples and in strawberry juice samples, by liquid chromatography with diode array detection (LC-DAD). Chromatographic coelution problems either because of unknown matrix interferences or because of the increase of organic modifier to reduce chromatographic analysis times are investigated. pH (4.25) and proportion of organic modifier in acetonitrile–water ratios (11:89, v/v) used as mobile phases have been optimized for separation of mixture of nine phenolic acids. Results obtained in the resolution and quantitation of phenolic acids in standards mixture samples and strawberry samples at two proportions of organic modifier (11:89 and 40:60 acetonitrile–water (v/v) ratios) show that the proposed MCR-ALS approach reduces analysis times and solvent expenses and improves determinations in case of strong coelution . Limits on the use of MCR-ALS are investigated in the analysis of phenolic acids in strawberry samples.

Determination of number of significant components and key variables using genetic algorithms in liquid chromatography-nuclear magnetic resonance spectroscopy and liquid chromatography-diode array detection

A new method is proposed for determination of the number of significant components in hyphenated chromatographic data. The method is based on the application of genetic algorithms (GA). The method is applied to five datasets from on-flow liquid chromatography with nuclear magnetic resonance spectroscopy (LC-NMR) and on three datasets from liquid chromatography with diode array detector (LC-DAD). The effect of different factors such as the application of technique on time or spectral direction, noise level, baseline, chromatographic resolution and relative peak height has been studied. The method is compared with other chemometric approaches for rank analysis and variable selection. The results produced by GA are promising on both types of data.

Analysis of three- and four-way data using multivariate curve resolution-alternating least squares with global multi-way kinetic fitting

This paper demonstrates a novel implementation of an alternating least squares (ALS) algorithm for resolving three- and four-way data. Computer-simulated multi-way data are studied as well as the multi-way data obtained from typical kinetic experiments observed using liquid chromatography with diode array detection (LC-DAD) and UV–visible spectroscopy. Each data set is analyzed using this new multi-way ALS algorithm, not only providing estimates of the spectral profiles (and retention profiles in the case of LC-DAD measurements) for each of the components involved, but also simultaneously estimating the rate constants for the reaction steps at different experimental conditions using a global kinetic analysis. However, when the reaction conditions do not require that all the rate constants are identical for each experiment, as is the case when the reactions are observed at different temperatures, the data analysis still benefits from the common information present in the data, such as spectral and retention profiles, as well as a common reaction mechanism.

Fast chromatography of complex biocide mixtures using diode array detection and multivariate curve resolution

Use of Multivariate Curve Resolution Alternating Least Squares (MCR-ALS) is evaluated in the analysis of complex biocide environmental sample mixtures by liquid chromatography with diode array detection (LC-DAD). Chromatographic coelution problems caused either because of the presence of unknown matrix interferences or because of using short chromatographic columns to reduce analysis times are investigated. Under such circumstances, lack of chromatographic resolution and lack of spectral selectivity of UV–VIS diode array detection is compensated by chemometric resolution using Multivariate Curve Resolution. Resolution of complex environmental mixtures and quantitative calibration curves for two types of chromatographic columns (25 and 7.5 cm) with different resolution and analysis times are shown. The limits of the proposed approach are investigated in the analysis of complex environmental samples with short LC columns and UV–VIS diode array detection.

Domoic acid accumulation in French shellfish in relation to toxic species of Pseudo-nitzschia multiseries and P. pseudodelicatissima

Within the French phytoplankton monitoring network (REPHY), domoic acid (DA), the toxin responsible for amnesic shellfish poisoning, was first detected in samples collected in 1998. Toxin analysis by the official method [liquid chromatography with diode array detection (LC/DAD)] was performed when Pseudo-nitzschia cell concentration was greater than 1.0×10 5 cells/l. LC/DAD results obtained in 1999 and 2000 showed increased DA accumulation in bivalves sampled at different sites along French coasts. The toxin maximum in 1999 was 3.2 μg DA/g of whole tissue, whereas the levels in 2000 (53 μg) were above the sanitary threshold (20 μg DA/g tissue). Phytoplankton samples collected during blooms were observed by both light and scanning electron microscopy (SEM). Identification of phytoplankton species by SEM analyses confirmed the presence of two known DA-producing species, P. pseudodelicatissima and P. multiseries. LC/DAD results for a mass culture of P. multiseries indicated that this species was involved in DA accumulation in French shellfish.

Evaluation of anti-pyrene and anti-fluorene immunosorbent clean-up for PAHs from sludge and sediment reference materials followed by liquid chromatography and diode array detection

The determination of polycyclic aromatic hydrocarbons (PAHs) in complex matrices such as sludges or sediments was performed by off-line enrichment using four different immunosorbents (IS). These IS are anti-fluorene and anti-pyrene antibodies immobilized on silica. The method involves the extraction of PAHs from water samples onto either two anti-fluorene or two anti-pyrene IS followed by analysis using liquid chromatography with diode array detection (LC-DAD) and its validation using gas chromatography coupled to a mass spectrometer (GC-MS). The procedure also uses a solubilizer to limit unwanted adsorption on vessels or tubing. In order to be able to evaluate the four IS, we have analyzed environmental sediments and sludge reference materials containing PAHs. Sediments were extracted by sonication with dichloromethane–methanol (2∶1) and the extracts were brought up to a volume of 100 mL of water in order to perform the extraction with the anti-fluorene and anti-pyrene IS. This method was optimized for 13 of 16 PAHs included in the Environmental Protection Agency US (EPA) priority list. The selectivity of the IS cleanup and the LC-DAD spectra allowed a good identification and quantification in the analysis of sediment and sludge complex samples containing the priority PAHs established by the EPA. The results obtained with the four IS were compared after determination of PAHs in complex sewage sludge and sediment reference matrices. Moreover, clean-up using IS was compared with conventional clean-up procedures and showed a better selectivity for PAHs by the IS procedures.

Persistence of selected pesticides and their phenolic transformation products in natural waters using off-line liquid solid extraction followed by liquid chromatographic techniques

The degradation of three organophosphorus pesticides with phenolic type structure (fenitrothion, ethyl-parathion, methylparathion) and pentachlorophenol in natural waters from Porto (Portugal) was studied. Three different types of natural waters (river water, estuarine water and ground water) were spiked with ethyl-parathion, methyl-parathion, fenitrothion and pentachlorophenol at 40 μg/l level and were exposed outdoor to ambient sunlight and temperature in natural conditions at the Faculty of Pharmacy of Porto during a period up to 7 weeks. Water samples were placed in 1 1 Pyrex flasks, properly closed. The samples were exposed day and night at these conditions during May and June 1996, the outside temperature being 22 °C with 5.5 h of sunlight exposition per day on average during the period studied. In order to monitor the degradation process, 100 ml of water was preconcentrated periodically by means of liquid solid extraction (LSE) using the polymeric sorbent Lichrolut EN from Merck (Darmstadt, Germany) and the extract was determined by liquid chromatography with diode array detection (LC-DAD). The use of LC coupled with mass spectrometry detection (LC-APcI-MS) permitted the unequivocal identification of various degradation products (4-nitrophenol, 3-methyl-4-nitrophenol, paraoxon ethyl, paroxon methyl, fenitrooxon and S-methylisomer of fenitrothion). Half-life for methyl-parathion was of 3 days in ground water and 4 days in both estuarine and river water; half-life for ethyl-parathion was of 2 days in ground and estuarine water and 3 days in river water; values of 1 day and less than 2 h were obtained for fenitrothion and pentachlorophenol, respectively, in all types of water. All transformation products resulted to be more stable than parent compounds except in ground water conditions. Half-lives of 5 and 4 days were calculated for 4-nitrophenol and paraoxon ethyl, respectively, in estuarine and river water. Values of 3 and 4 days were obtained for 3-methyl-4-nitrophenol in estuarine and river water, respectively.

Alternative Algorithm for Simultaneous Determinations of Components Poorly Resolved by Liquid Chromatography with Multiwavelength Detection.

This paper describes an alternative algorithm for a simultaneous multicomponent analysis with three-mode three-dimensional array data generated by liquid chromatography with diode array detection (LC-DAD). The algorithm is mainly based on a combination of two-step Moore-Penrose pseudoinverse computations based on a truncated singular value decomposition (TSVD) and one-step direct bilinear decomposition. It is modeled with a series of pure or mixed reference samples and then used to analyze several unknown samples. The algorithm was evaluated by Monte-Carlo simulations of two synthetic LC-DAD data sets with different noise levels, and applied to the simultaneous determination of chlorobenzene and toluene by means of HPLC with a photodiode array detector. The obtained results show that the proposed algorithm is suitable for simultaneous determinations of several components, of which the chromatographic peaks and absorption spectra heavily overlap each other, provided that their three-dimensional data have trihnear properties.

Corrections for heteroscedasticity in window evolving factor analysis

Window evolving factor analysis (WEFA) is a powerful technique for checking peak purity in liquid chromatography with diode array detection (LC-DAD). However, practical application of the technique can be limited by instrumental and experimental non-idealities. One of the problem sources is heteroscedasticity. In this work, we propose two new data transformation procedures and one technique for directly adjusting the log-eigenvalue profiles, which eliminate most of the heteroscedastic effect in the WEFA plots. The pretreatment and the adjustment techniques can be used in combination to obtain even better results. The performance of the techniques are demonstrated on simulated and actual data.

Validation of an automated precolumn exchange system (PROSPEKT) coupled to liquid chromatography with diode array detection. Application to the determination of pesticides in natural waters

An automated precolumn exchange system (PROSPKET) coupled to liquid chromatography with diode array detection (LC-DAD) has been used for the determination of trace pesticides in natural (drinking and ground) waters. Relevant parameters such as pH and type of precolumn (C 18 and PLRP-s) were optimized for a variety of organophosphorus pesticides, herbicides and pesticide transformation products. Calibration graphs at low levels of determination (0.1–1.5 μg/l) were constructed. The validation of the present automated method has been guaranteed by participating in Aquacheck interlaboratory exercises where more conventional gas chromatographic determinations are being used. The overall relative standard deviation between values obtained in our laboratory and the average value obtained by 14–15 other laboratories varied between 1.6 and 36% for most of the studied compounds. Problems encountered in the determination of certain organophosphorus pesticides led to high variations in the mean values and errors of many laboratories. In this sense, problematic compounds like mevinphos, which gave two peaks corresponding to the cis- and trans-isomers, parathion-methyl and diazinon, which showed coelution problems, or malathion, which presented poor quantitation due to low absorption in the UV range. In addition, fenthion, which was not determined gave 3–4 compounds, corresponding to various degradation products. Confirmation of the different pesticides exhibiting an UV maximum > 220 nm was feasible due to their characteristic UV spectra and satisfactory levels of 0.02 μg/l were found when preconcentrating 150 ml of ground water sample.