Photodiode Array Detection System Research Articles

High throughput analytical approach based on μQuEChERS combined with UHPLC-PDA for analysis of bioactive secondary metabolites in edible flowers

Mallow blue (Malva sylvestris L.), hibiscus (Hibiscus rosa-sinensis L.) and nasturtium (Tropaeolum majus L.), are common edible flowers rich in bioactive secondary metabolites (BASMs) whose use in sophisticated gastronomy present currently as increasing trend. In this study the BASMs profile of these edible flowers was established using an emerging green extraction technique, μQuEChERS followed by ultra-high performance liquid chromatography coupled to a photodiode array detection system (UHPLC-PDA). After validation the μQuEChERS/UHPLC-PDA methodology allow to identify that apigenin and epigallocatechin gallate are the most abundant BASMs in mallow blue flowers, while catechin and dicaffeoylquinic acid are predominant in hibiscus flowers, and myricitrin and dicaffeoylquinic acid in nasturtium flowers. Total polyphenol content is the highest in the extract of hibiscus. Nasturtium shows the greatest radical scavenging activity. The results revealed that these flowers constitute a potential source of BASMs with different bioactive properties suggesting its use in design of new functional foods.

µQuEChERS Combined with UHPLC-PDA as a State-of-the-Art Analytical Approach for Quantification of Chlorpropham in Potato

Pesticides are chemicals used in agriculture to prevent insects, fungi, weeds, and other pests, from damaging crops. In addition, some types of pesticides are used after harvest as sprout suppressant agents help keeping the quality parameters of crops during storage. Nonetheless, its presence, even at trace levels, in food products is becoming a big challenge regarding human health. The current work aimed to develop and validate a sensitive and high-throughput analytical approach, based on a state-of-the-art microextraction technique—µQuEChERS, combined with ultra-high performance liquid chromatography equipped with a photodiode array detection system (UHPLC-PDA) to quantify isopropyl-N-(3-chlorophenyl) carbamate (chlorpropham), commonly used as efficient sprout suppressant stored potatoes, in raw and cooked potatoes cultivated in different geographical regions of Madeira Island (Portugal). Good results were obtained in terms of figures of the merit of the method, with correlation coefficients (R2) higher than 0.999 and recoveries between 94.5% to 125%. Method limit of detection (LOD) and limit of quantification (LOQ) were 0.14 μg/Kg and 0.43 μg/Kg, respectively, which are much lower than the accepted and legislated requirements by the European Union, which is 20 μg/Kg for chlorpropham. The concentration of chlorpropham in raw potatoes is significantly higher when compared to cooked samples, which revealed that the thermic treatment during cooking had a significant effect on its degradation. A significant decrease (90%, on average) was observed on chlorpropham levels.

Chemical Fingerprint of Free Polyphenols and Antioxidant Activity in Dietary Fruits and Vegetables Using a Non-Targeted Approach Based on QuEChERS Ultrasound-Assisted Extraction Combined with UHPLC-PDA.

Fruits and vegetables are considered a good source of antioxidants, which are beneficial in protecting the human body against damage induced by free radicals and other reactive oxygen (ROS) and nitrogen (RNS) species. In this work, we aimed to evaluate the integral antioxidant activity (AOA) and determine individual polyphenols in fruits and vegetables of frequent consumption. For this purpose, an innovative and high throughput analytical approach based on original QuEChERS assisted by ultrasound extraction (USAE), instead of the manual agitation used in the classical procedure, was optimized and implemented for the isolation of polyphenols. The total phenolic content (TPC), flavonoids, anthocyanins, and betalains were evaluated using different spectrophotometric assays. In addition, free radical scavenging by methods 2,2’-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,2′-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) were used to estimate the AOA of the investigated fruit and vegetable extracts. Red onion, tamarillo, and beetroot were the samples with the highest AOA. The quantification and identification of free low molecular weight polyphenols from QuEChERS-USAE extracts was carried out by ultra-high-pressure liquid chromatography equipped with a photodiode array detection system (UHPLC-PDA). Catechin was the most abundant polyphenol, followed by gentisic and ferulic acids, mainly in the watercress sample. In relation to flavonols, quercetin and kaempferol were found mostly in onion samples, and in small quantities in tomato and watercress. The improved analytical approach, QuEChERS-USAE/UHPLC-PDA, offers an attractive alternative for the analysis of polyphenols from fruit and vegetable samples, providing several advantages over traditional extraction techniques, in terms of reproducibility, simplicity, low cost, analysis speed, and analytical performance.

The use of a microwave-assisted solvent extraction coupled with HPLC-UV/PAD to assess the quality of Marrubium vulgare L. (white horehound) herbal raw material.

Marrubium vulgare is a herbal remedy presents in several European Pharmacopoeias and commonly marketed as white horehound. The chemotaxonomic marker of Marrubium genus is marrubiin and its content may change in response to biotic and abiotic stress. Development of a microwave-assisted solvent extraction (MASE) methodology suitable for exhaustively extracting marrubiin from M. vulgare leaves, easily applicable to large sets of samples. Evaluation of the influence of copper(II) on marrubiin production. M. vulgare leaves were dried, extracted exploiting MASE and analysed via high-performance liquid chromatography ultraviolet photodiode array detection (HPLC-UV/PAD) system. A design of experiments approach was adopted to select the best extraction conditions. Extraction parameters (solvent composition, extraction time and temperature), were studied applying two full factorial experimental designs in a sequential approach. To analyse samples, a rapid HPLC-UV/PAD method was set up. The best results in terms of marrubiin extraction yield were obtained extracting samples at 120°C with 100% ethanol, for 15min (3 × 5 min microwave cycles). The developed methodology was successfully applied to matrices grown in Greenhouse conditions and under stress induced by copper(II), selected as model agent for abiotic stress. Progressively decreasing production of marrubiin was evidenced in connection with treatment with 80, 200 and 300mg/L copper sulphate. An efficient methodology for the extraction and determination of the amount of marrubiin in large sets of samples of M. vulgare plants was developed. Results demonstrated that marrubiin is an easily detectable marker useful for evaluating M. vulgare reaction to stress.

An improved and miniaturized analytical strategy based on μ-QuEChERS for isolation of polyphenols. A powerful approach for quality control of baby foods

An improved and miniaturized quick, easy, cheap, effective, rugged and safe (μ-QuEChERS) strategy combined with ultra-high pressure liquid chromatography coupled to a photodiode array detection system (UHPLC-PDA) was developed and optimized for the determination of twelve polyphenols in different baby foods samples. The proposed analytical approach proved to be highly cost-effective and environmentally friendly, since it showed good extraction efficiency using few amounts of sample (0.3g), organic solvents (1000μL), clean-up sorbents (87.5mg) and partitioning salts (0.2g), producing minimal waste disposal and reducing analysis time. Method performance was validated in terms of selectivity, linearity, limits of detection and quantification, matrix effects, precision and accuracy using different baby food matrices according to their composition. The overall average recoveries ranged from 71 to 100% with relative standard deviations lower than 6% (n=18). The method was successfully applied to commercial baby food samples (including pureed solids, juices and porridges), and their total phenolic content (TPC) and antioxidant capacity (RSA) were also investigated. Results allowed characterizing the abundance of the selected polyphenols in the samples, and the highest RSA and TPC were found in the fruit-based baby foods. Thus, this work represents a first approach to determine the nutritional quality of these products.

The Degradation Capability of Bacillus alkalinitrilicus for Imidacloprid Bioremediation in Soil

Biodegradation of imidacloprid is reported in sterile sandy loam soil fortified @ 50, 100 and 150 mg kg−1 insecticide along with 45x107 colony forming unit (cfu) of Bacillus alkalinitrilicus. Soil samples were analyzed against those treated with the same doses of the insecticide but without inoculating the bacteria. The residues of imidacloprid and its metabolites were determined by high performance liquid chromatography employing photodiode array detector system and C18 column. After 7 d of treatment, the total residues in soil treated with 50 mg kg−1 imidacloprid were 40.30 mg kg−1, compared with 36.01 mg kg−1 in presence of bacteria. On the same day, the metabolites like 6-chloronicotinic acid, nitroguanidine, olefine, nitrosimine, urea and 5-hydroxy were detected in all the treatments. Among metabolites, urea, olefine and 6-chloronicotinic acid were found in major proportion while 5-hydroxy, nitrosimine and nitroguanidine were observed in minute amounts. The half-life of imidacloprid at 50 mg kg−1 dose was 25 d and 30 d for both the 100 and 150 mg kg−1 doses in the unamended soil. However, in amended soil, the half-life at 50 and 100 mg kg−1 were 15 d and at 150 mg kg−1, it was 17 d. B. alkalinitrilicus can thus be used for bioremediation of imidacloprid in soil.

Optical attenuator for Geiger mode avalanche photodiode laser detection systems

This paper presents a method to estimate the optical attenuator according to the false firing percentage of Geiger Mode Avalanche Photodiode array detection system only under background light. The effect of the dark current and the gate setting are discussed. The efficiency of the Geiger mode avalanche photodiode detection system is improved by the optical attenuator by simulation analysis, that the false probability is reduced in all cases, and the detection probability increases accorindgly when the signal is larger than the total noise. Finally, the imaging simulation experiments show that the average ranging accuracy increase by using optical attenuator.

Development and Validation of an HPLC Method for Determination of Thiamethoxam and Its Metabolites in Cotton Leaves and Soil.

An easy and simple analytical method was standardized and validated for the estimation of residues of thiamethoxam and its metabolites in cotton. The samples were extracted with acetonitrile, water, and methanol; diluted with brine solution; partitioned into dichloromethane and ethyl acetate; dried over anhydrous sodium sulfate; and cleaned up by glass column chromatography. Final clear extracts were concentrated under vacuum and reconstituted into HPLC grade acetonitrile, and residues were estimated using an HPLC instrument equipped with a C18 column and photodiode array detector system. Acetonitrile-1% formic acid in HPLC grade water (30 + 70) was used as mobile phase at 0.2 mL/min. Consistent recoveries ranging from 82 to 97% for thiamethoxam and its metabolites were observed when samples were spiked at 0.05-1.0 mg/kg levels. The LOQ of the method was determined to be 0.05 mg/kg. The analytical method was validated in terms of the selectivity, linearity, precision, and accuracy of the detection system.

Erratum for: Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02 % trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2 % to 20 %, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Supercritical Fluid Chromatography as an Alternative Tool for the Qualitative and Quantitative Analysis of Metarhizium brunneum Metabolites from Culture Broth

A fast and selective ultrahigh-performance supercritical fluid chromatography photodiode array detector method was established for the qualitative and quantitative analysis of destruxins, cyclic hexadepsipeptides, from fungal culture broth samples. Prior to analysis, sample purification was carried out using an off-line solid-phase extraction protocol on a reversed-phase material in order to remove unwanted matrix constituents. For separation, detection, and identification, an ultrahigh-performance supercritical fluid chromatography photodiode array detector system hyphenated to a triple quadrupole mass spectrometer was utilized. Analyses were performed on an Acquity ethylene bridged hybrid 2-ethylpyridine sub 2 µm particle size column with CO2 and an acidified (0.02% trifluor acetic acid) modifier mixture of methanol/acetonitrile (8/2 v/v) serving as mobile phase. For the optimal separation of destruxins, the amount of the modifier was increased in a 10 min linear gradient from 2% to 20%, and the column outlet pressure and temperature was set at 140 bars and 60 °C, respectively. Seventeen analytes were separated within an elution window of 4 minutes. Five destruxin congeners (destruxin A, destruxin B, destruxin D, destruxin E, and destruxin E-diol) were identified using reference material. Additionally, eight analytes were tentatively assigned as known destruxins by the evaluation of mass spectrometry data performed as multiple reaction monitoring experiments in the positive electrospray ionization mode.

Development and validation of a quick, easy, cheap, effective, rugged and safe method for the determination of imidacloprid and its metabolites in soil.

A simplified quick, easy, cheap, effective, rugged and safe method was standardized and validated for the estimation of residues of imidacloprid and its metabolites from different types of soil comprising sandy loam, loamy sand and clay loam soil. The samples were extracted with acetonitrile, clean up by treatment with primary secondary amine sorbent and graphitized carbon. High-performance liquid chromatography (HPLC) is a technique to separate and quantitate residues of polar, nonvolatile and thermolabile chemical compounds, using high-pressure pumps, short and narrow columns packed with microparticulate phases and a detector. The residues were estimated using HPLC equipped with a photodiode array detector system, C18 column. Acetonitrile and water (30 : 70) were used as an isocratic mobile phase at 0.3 mL/min. Imidacloprid and its metabolites presented distinct peaks at retention factors of 4.93 min (6-chloronicotinic acid), 7.91 min (nitroguanidine), 9.12 min (olefin), 11.32 min (nitrosimine), 13.82 min (urea), 15.45 min (5-hydroxy) and 22.47 min (imidacloprid). Consistent recoveries above 80% for imidacloprid and its metabolites were observed when samples were spiked at 0.01, 0.05 and 0.10 mg/kg levels. The limit of quantification of the method was 0.01 mg/kg. The analytical method was validated in terms of parameters including selectivity, linearity, precision and accuracy of the detection system.

Sensitive methodology for simultaneous determination of residues of imidacloprid and its metabolites in sugarcane leaves and soil.

An analytical method to quantify imidacloprid and its metabolites in sugarcane leaves and soil using HPLC has been developed. The samples were extracted with acetonitrile + water (80 + 20, v/v), soil samples partitioned with dichloromethane, and leaf samples with hexane + ethyl acetate (9 + 1, v/v) and dichloromethane. Further, the extracts were dried, filtered, and concentrated under vacuum into HPLC-grade acetonitrile. Residues were estimated using an HPLC equipped with a photodiode array detector system, C18 column with a mobile phase of acetonitrile + water (40 + 60, v/v) at 0.3 mL/min to separate imidacloprid and its six metabolites in single run of 20 min. The mean percent recoveries of imidacloprid and its metabolites ranged from 80.45 to 99.80 from sugarcane leaves and 80.20 to 99.70 from sugarcane soils. The analytical method was validated in terms of selectivity, linearity, reproducibility, repeatability, and accuracy. The repeatability values ranged from 0.24 to 3.15% and 1.69 to 4.94%, along with 2.73 to 3.82% and 1.12 to 4.96%, for imidacloprid and its metabolites in leaves and soil, respectively. The reproducibility of imidacloprid and its metabolites in leaves and soil ranged from 2.20 to 4.27% and 2.53 to 4.08%, respectively, and all measurements were within 15% at all concentration levels.

A fast method using a new hydrophilic–lipophilic balanced sorbent in combination with ultra-high performance liquid chromatography for quantification of significant bioactive metabolites in wines

This manuscript describes the development and validation of an ultra-fast, efficient, and high throughput analytical method based on ultra-high performance liquid chromatography (UHPLC) equipped with a photodiode array (PDA) detection system, for the simultaneous analysis of fifteen bioactive metabolites: gallic acid, protocatechuic acid, (−)-catechin, gentisic acid, (−)-epicatechin, syringic acid, p-coumaric acid, ferulic acid, m-coumaric acid, rutin, trans-resveratrol, myricetin, quercetin, cinnamic acid and kaempferol, in wines. A 50-mm column packed with 1.7-μm particles operating at elevated pressure (UHPLC strategy) was selected to attain ultra-fast analysis and highly efficient separations. In order to reduce the complexity of wine extract and improve the recovery efficiency, a reverse-phase solid-phase extraction (SPE) procedure using as sorbent a new macroporous copolymer made from a balanced ratio of two monomers, the lipophilic divinylbenzene and the hydrophilic N-vinylpyrrolidone (Oasis™ HLB), was performed prior to UHPLC–PDA analysis. The calibration curves of bioactive metabolites showed good linearity within the established range. Limits of detection (LOD) and quantification (LOQ) ranged from 0.006μgmL−1 to 0.58μgmL−1, and from 0.019μgmL−1 to 1.94μgmL−1, for gallic and gentisic acids, respectively. The average recoveries±SD for the three levels of concentration tested (n=9) in red and white wines were, respectively, 89±3% and 90±2%. The repeatability expressed as relative standard deviation (RSD) was below 10% for all the metabolites assayed. The validated method was then applied to red and white wines from different geographical origins (Azores, Canary and Madeira Islands). The most abundant component in the analysed red wines was (−)-epicatechin followed by (−)-catechin and rutin, whereas in white wines syringic and p-coumaric acids were found the major phenolic metabolites.The method was completely validated, providing a sensitive analysis for bioactive phenolic metabolites detection and showing satisfactory data for all the parameters tested. Moreover, was revealed as an ultra-fast approach allowing the separation of the fifteen bioactive metabolites investigated with high resolution power within 5min.

Comparative application of microwave, ultrasonication, ultracentrifugation and conventional heating for preparation of sample as dinitrophenyl derivative for direct enantioseparation of certain amino alcohols and 1-amino-2-propanol from vitamin B 12 hydrolysate on α 1-acid glycoprotein and β-cyclodextrin columns

Taking into account the structural similarities of amino alcohols with amino acids and in order to reduce time for derivatization unconventional approaches viz. microwave irradiation, ultrasonication and ultra centrifugation were applied for synthesis of dinitrophenyl derivatives of nine amino alcohols and to work out a method of choice for sample preparation for direct enantioseparation. The enantiomeric dinitrophenyl derivatives so synthesized were separated on α 1-acid glycoprotein and β-cyclodextrin columns with detection at 230 nm using photodiode array detection system. Derivatization methods and chromatographic parameters were optimized. β-Cyclodextrin column was found better compared to AGP column for enantioseparation. Limit of detection, quantification, accuracy and precision were also determined. The developed method was successfully applied to determine enantiomeric purity of 1-amino-2-propanol obtained from vitamin B 12 hydrolysate.

Photodecomposition of o-phthaldialdehyde-derivatized amino acids by the photodiode array detector during their high-performance liquid chromatographic analysis

During the high-performance liquid chromatographic (HPLC) analysis of o-phthaldialdehyde (OPA)-derivatized amino acids such as arginine and homoarginine, we observed that the response of the fluorescence detection (FLD) system is decreased when the photodiode array detection (DAD) system (located before the FLD system) was simultaneously switched on. The decrease in the FLD response, i.e., the difference in the FLD peak area (Δ A) obtained with DAD modes off ( A off) and on ( A on) was dependent upon the flow rate, but the relative FLD response decrease (Δ A/ A off) was practically independent of the amount of analyte injected. For example, decreasing the flow rate from 1 to 0.5 mL/min resulted in the relative decrease of FLD response from ∼5% to 11%. It was concluded that DAD caused a photoinduced partial decomposition of the OPA-derivatized amino acids flowing through the cell. The photoinduced dissociation of OPA derivatives was independently supported by using off-line photodiode array spectrometric measurements with long and short irradiation pulses. Based on the experimental results, for description of the variation of FLD responses with the flow rate upon the irradiation by DAD a simple mathematical model is proposed and reported.

Comprehensive normal-phase × reversed-phase liquid chromatography coupled to photodiode array and mass spectrometry detection for the analysis of free carotenoids and carotenoid esters from mandarin

In the present work, a novel strategy including the use of two different comprehensive HPLC methods has been employed to study the whole carotenoid composition of mandarin essential oil. Thus, two different fully orthogonal two-dimensional HPLC methods have been used. A silica microbore column was coupled to a C 18 monolithic column to study the mandarin saponified extract, while the coupling of a cyano microbore column to a C 18 monolithic column was employed to study the intact mandarin essential oil sample in order to characterize the native carotenoid esters composition. Detection was performed by connecting a photodiode array detection (DAD) system in parallel with a MS detection system operated with an atmospheric pressure chemical ionization (APCI) interface. Thus, the carotenoid identification was carried out by combining the information provided by the DAD and MS systems and the peaks relative position in the two-dimensional chromatograms.

Phenolic Profile of Asturian (Spain) Natural Cider

The polyphenolic composition of natural ciders from the Asturian community (Spain), during 2 consecutive years, was analyzed by RP-HPLC and the photodiode-array detection system, without previous extraction (direct injection). A total of 16 phenolic compounds (catechol, tyrosol, protocatechuic acid, hydrocaffeic acid, chlorogenic acid, hydrocoumaric acid, ferulic acid, (-)-epicatechin, (+)-catechin, procyanidins B2 and B5, phloretin-2'-xyloglucoside, phloridzin, hyperin, avicularin, and quercitrin) were identified and quantified. A fourth quercetin derivative, one dihydrochalcone-related compound, two unknown procyanidins, three hydroxycinnamic derivatives, and two unknown compounds were also found. Among the low-molecular-mass polyphenols analyzed, hydrocaffeic acid was the most abundant compound, representing more than 80% of the total polyphenolic acids. Procyanidins were the most important family among the flavonoid compounds. Discriminant analysis was allowed to correctly classify more than 93% of the ciders, according to the harvest year; the most discriminant variables were an unknown procyanidin and quercitrin.

Liquid chromatographic method for quantifying polyphenols in ciders by direct injection

An analytical method for the quantitative determination of the principal phenolic compounds (benzoic acids, hydroxycinnamic acids, 3-phenylpropionic acids, flavanols, procyanidins, dihydrochalcones, quercetin glycosides) in ciders, which successfully employs a RP-HPLC and photodiode-array detection system without prior treatment of the sample, is described. Parameters usually examined in the method validation were evaluated. Good linearity was obtained with correlation coefficients exceeding 0.999 and the detection limits ranged from 0.07 mg/L ( p-hydroxybenzoic acid) to 2 mg/L (hydrocaffeic acid). Recoveries ranging between 90 and 104% and the reproducibility of the method was always <8% (RSD). The method was applied to a set of commercial samples and the results obtained may be helpful to establish a phenolic profile in Asturian cider.

Hypericin and Pseudohypericin. Purity Criteria and Quantitative Determination in Extracts of Saint John’s Wort (Hypericum perforatum)

Hypericin and pseudohypericin in extracts of St. John’s Wort (Hypericum perforatum) are assayed using UV-Vis, measuring the absorbance at 588 nm of a methanolic solution of the compounds. The value of the absorption coefficient is difficult to define insofar as none of the routine purity criteria can be applied to these two compounds. Analysis of the entire UV-Vis spectrum from 220 to 740 nm would seem to be a reliable way of comparing different samples. A UV-Vis spectrum was recorded for each of the compounds using a Photodiode array detection system. A more accurate way of determining the levels of hypericin and pseudohypericin in extracts of St. John’s Wort using these reference spectra is described.

Determination of difloxacin and sarafloxacin in chicken muscle using solid-phase extraction and capillary electrophoresis

This paper describes a method for residue analysis of difloxacin and sarafloxacin in chicken muscle. Clean-up and preconcentration of the samples are effected by solid-phase extraction (C 18) and the determination is carried out by capillary electrophoresis using a photodiode array detection system. The method was validated with satisfying results. The calibration graphs are linear for difloxacin and sarafloxacin from 50 to 300 μg/kg. The limit of detection obtained for difloxacin and sarafloxacin are 10 and 25 μg/kg, respectively, which allows the detection of positive muscle samples at the required maximum residue limits of European Union.