Post-column Photochemical Reactor Research Articles

Chromatographic Analysis of Aflatoxigenic Aspergillus flavus Isolated from Malaysian Sweet Corn

High-performance liquid chromatography (HPLC) provides a quick and efficient tool for accurately characterizing aflatoxigenic and non-aflatoxigenic isolates of Aspergillus flavus. This method also provides a quantitative analysis of AFs in Aspergillus flavus. The method’s recovery was assessed by spiking a mixture of AF at different concentrations to the testing medium. The validity of the method was confirmed using aflatoxigenic and non-aflatoxigenic strains of A. flavus. The HPLC system, coupled with a fluorescence detector and post-column photochemical reactor, showed high sensitivity in detecting spiked AFs or AFs produced by A. flavus isolates. Recovery from medium spiked with 10, 20, 60, and 80 ppb of AFs was found to be 73–86% using this approach. For AFB1 and AFB2, the limit of detection was 0.072 and 0.062 ppb, while the limit of quantification was 0.220 and 0.189 ppb, respectively. The AFB1 concentrations ranged from 0.09 to 50.68 ppb, while the AFB2 concentrations ranged between 0.33 and 9.23 ppb. The findings showed that six isolates produced more AFB1 and AFB2 than the acceptable limit of 5 ppb. The incidence of aflatoxigenic isolates of A. flavus in sweet corn and higher concentrations of AFB1 and AFB2 emphasize the need for field trials to explore their real potential for AF production in corn.

Aflatoxin in raw peanut kernels marketed in Malaysia

The occurrence of aflatoxin in eighty-four samples of raw peanut kernels which are randomly collected from Malaysian supermarkets was examined. Analysis for aflatoxin was performed by solvent extraction and immunoaffinity clean-up followed by the determination using high performance liquid chromatography equipped with post-column photochemical reactor for enhanced detection and fluorescence detector. A detection limit of 0.01-0.09 ng/mL and a quantification limit of 0.04-0.30 ng/mL were obtained. The aflatoxin concentrations ranged from not detected to 97.28 ng/g in all samples investigated. About 78.57% of the samples were contaminated with aflatoxin, of which 10.71% exceeded the maximum tolerable limit of 15 ng/g set by the Codex. Average recoveries of the aflatoxin analysis were acceptable which were in the range of 74.85 ± 8.83% for AFG2 at the concentration of 0.15 ng/mL and 103.91 ± 6.45% for AFB2 at the concentration of 0.15 ng/mL. The average daily intake estimated for total aflatoxins was 10.69 ng/kg body weight. There was a significant difference (P < 0.05) in aflatoxin content between brands and locations.

An aptamer affinity column for purification and enrichment of aflatoxin B1 and aflatoxin B2 in agro-products.

We have developed an aptamer affinity column (AAC) for the purification and enrichment of trace aflatoxin B1 (AFB1) and aflatoxin B2 (AFB2) in genuine agro-products through the covalent conjugation of amino modified aptamer and NHS-activated Sepharose. The coupling and working conditions found to be suitable for this AFB-AAC were examined in regard to coupling time (2min), loading volume (30mL), and the methanol concentration (< 10%) used in the loading step. The performance of AFB-AAC was then further evaluated in terms of capacity (329.1 ± 13.7ng for AFB1 and 162.5 ± 8.9ng for AFB2), selectivity (excellent), reusability (twenty-three times for AFB1 and twelve times for AFB2), and repeatability (92.7% ± 2.9% for AFB1 and 71.5% ± 3.4% for AFB2). Furthermore, the AAC clean-up combined with HPLC-FLD demonstrated excellent linearity over a wide range, good sensitivity with an LOD of 50pgmL-1 for AFB1 and 15pgmL-1 for AFB2, and acceptable recovery with different spiking levels in different matrices. Finally, the AAC was successfully applied to analyte AFB1 and AFB2 in four types of agro-products as well as a maize flour reference material, and the results were found to be in accordance with those of commercial IACs. This study provides a reference for the analysis of other trace analytes by merely changing the corresponding aptamer and represents a strong contender for immune affinity columns. Graphical abstract An aptamer affinity column for purification and enrichment of aflatoxin B1 and aflatoxin B2 in agro-products with the aid of HPLC-FLD and a post-column photochemical derivatization reactor.

A sensitive photochemical reaction-capacitively coupled contactless conductivity detection system for HPLC and its application in determination of Cyclosporin A

High performance liquid chromatography (HPLC) post-column photochemical reaction (PR) coupled capacitively coupled contactless conductivity detector (C4D) was used for the first time in analysis of weak ultraviolet (UV)-absorbing, non-fluorescence and nonpolar compound. A series of conditions including the radiation power of light source, the length of the reaction tube and the thickness of detection tube were investigated. HPLC-PR-C4D system was successfully applied to the determination of Cyclosporin A (CsA). Consequently, under optimal conditions, the detection system exhibited a detection limit of 0.04 μg/mL and wide linear range from 0.5 μg/mL to 100 μg/mL for CsA detection. Application of the HPLC-PR- C4D system to pharmaceutical formulation and biological samples revealed the system developed maybe reliably applied to clinical studies.

Determination of nitenpyram and 6-chloronicotinic acid in environmental samples by ion chromatography coupled with online photochemically induced fluorescence detector.

A simple, cost-effective, sensitive, and quick method for the determination of nitenpyram and its metabolite 6-chloronicotinic acid in environmental samples was developed by coupling an ion chromatograph with a fluorescence detector and a post-column photochemical reactor. This developed analytical method involved a rapid sample extraction by modified and miniaturized quick, easy, cheap, effective, rugged, and safe method followed by isocratic ion chromatographic separation of nitenpyram and 6-chloronicotinic acid into an IonPac™ AS11-HC column protected by IonPac™ AG11A guard column by running 30mM NaOH + 10% acetonitrile mobile phase. A homemade post-column photochemical reactor was also integrated with the ion chromatographic system for online transformation of both analytes into their respective highly fluorescent photoproduct in basic media without using an extra pump. The developed method was validated by following SANTE/11945/2015 guidelines on analytical quality control and validation procedures. The method showed a good linear response (r>0.999), improved limit of detection (0.101-0.132μg/L), minimum or no matrix effect, excellent recoveries (90.2-100.10%) and relative standard deviations were found to be ≤6.50%.

HPLC-DAD-FLD Method for Simultaneous Determination of Mycotoxins in Wheat Bran.

Aflatoxins, deoxynivalenol, ochratoxin A and zearalenone are the most important mycotoxins that everyone on its own, in groups or simultaneously contaminate cereals. The external layers of cereal grains (bran) apart from health promoting ingredients are also the most contaminated part with reference to mycotoxin's presence. Therefore, consumption of a high fiber wheat-based diet represent an increased risk to consumer's health. The objective of this study was to develop a simple and reliable high performance liquid chromatography method for the simultaneous determination of these mycotoxins in wheat bran (WB). A double extraction was applied with phosphate buffered saline/methanol and for the clean-up a multi-immunoaffinity column was utilized. The detection was carried out with diode-array and fluorescence detectors linked with a post-column photochemical reactor. After optimization of the chromatographic conditions, all mycotoxins were eluted within ~26 min. Limits of detection for each mycotoxin (0.12-12.58 µg/kg) were below the maximum levels provisioned by European Union regulations. Good linearity was observed for the analytes (r2 ≥ 0.9980). The recovery of analyzed mycotoxins ranged from 70.2 to 105.8%, with a relative standard deviation <12%. The method was successfully applied to quantify mycotoxins in 34 WB samples obtained after pearling of grains that were collected from different regions of Greece.

Simultaneous determination of imidacloprid and carbendazim in water samples by ion chromatography with fluorescence detector and post-column photochemical reactor

A new analytical method has been developed and validated for the simultaneous determination of pesticides from different classes using ion chromatography-online photochemical derivatisation-fluorescence detector (IC-hv-FD). Fluorimetric detection was performed at λex/λem=332nm/367nm for imidacloprid and then detector was set at λex/λem=247nm/470nm for carbendazim. The two pesticides imidacloprid and carbendazim were successfully separated isocratically on an IonPac® AS11 (250mm×4mm i.d; 13µm particle size, Dionex) anion-exchange column using 40mM KOH with 10% (v/v) acetonitrile and pumped at a flow rate of 1.0mLmin−1. Under the optimized conditions, the limit of detection (LOD, S/N=3) of imidacloprid and carbendazim were 7.8µgL−1 and 67µgL−1, respectively. The experimental results showed that there was good linearity with a correlation coefficient (r)≥0.9966 over the range of 0.05–10mgL−1 for imidacloprid and 0.2–15mgL−1 for carbendazim. Good reproducibility with a relative standard deviation (RSD, n=7) less than 4.5%. Finally, the proposed method was applied with satisfactory results to the analysis of these pesticides in ground water, lake water and river water without any pre-treatment of samples. The average spiked recoveries were in the range of 90–104%.

Analysis of cocoa products for ochratoxin A and aflatoxins

Eighty-five samples of cocoa products sampled in Canada were analysed for ochratoxin A (OTA) and aflatoxins in 2011–2012. Inclusion of the aflatoxins in this survey required additional method development. Chocolate was extracted with methanol–water plus NaCl, while for cocoa two successive extractions with methanol and methanol–water were made. Extracts were cleaned on an AflaOchra immunoaffinity column (IAC). Determination was by reversed phase high performance liquid chromatography (HPLC). Detection of the aflatoxins was with a post-column photochemical reactor and of OTA by fluorescence detection. Mean limits of quantification (LOQ) of chocolate and cocoa powders were 0.16 ng/g (OTA) and 0.07 ng/g (aflatoxin B1), respectively. Survey results showed that the incidences of OTA above the LOQ in natural cocoa were 15/15 (mean 1.17 ng/g), 20/21 for alkalized cocoa (mean 1.06 ng/g), 9/9 for baking chocolate (mean 0.49 ng/g), 20/20 for dark chocolate (mean 0.39 ng/g), 7/10 for milk chocolate (mean 0.19 ng/g), 5/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. These results confirm our previous work with OTA. In the same samples, incidences of aflatoxin B1 above the LOQ were 14/15 for natural cocoa (mean 0.86 ng/g), 20/21 for alkalized cocoa (mean 0.37 ng/g), 7/9 for baking chocolate (mean 0.22 ng/g), 16/20 for dark chocolate (mean 0.19 ng/g), 7/10 for milk chocolate (mean 0.09 ng/g), 4/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. Both aflatoxins and OTA were confirmed by HPLC-MS/MS when OTA or aflatoxin levels found were above 2 ng/g in cocoa.

Fluorometric Determination of Nalidixic Acid in Human Urine Using High-Performance Liquid Chromatography with Postcolumn Photochemical Reaction

Fluorometric Determination of Nalidixic Acid in Human Urine Using High-Performance Liquid Chromatography with Postcolumn Photochemical Reaction

Detection of Aspergillus spp. and determination of the levels of aflatoxin B1 in rice imported to Bushehr, Iran

Aflatoxins (B 1 , B 2 , G 1 and G 2 ) are hepatotoxic metabolites produced by Aspergillus flavus on a number of agricultural commodities. Their levels were studied in rice samples imported to Iran through a southern port in Bushehr. Aflatoxins analysis was performed by solvent extraction, immunoaffinity clean-up and determination using high performance liquid chromatography (HPLC) equipped with post-column photochemical reactor and fluorescence detector. Among 152 samples analyzed, 75% showed levels of aflatoxin B1 (AFB1) contamination. However, there was no sample with AFB 1 above maximum tolerated level (MTL) of 5 ng/g assigned by Institute of Standard and Industrial Research of Iran (ISIRI). AFB 1 concentrations in the samples was 0.09 to 3.3 ng/g. Out of the 152 samples analyzed, about 76.97% were contaminated with total aflatoxin (AFT) with the mean of 0.671 ng/g which is lower than MTL of AFT in rice (30 ng/g) assigned by (ISIRI). AFT concentration ranged from 0.15 to 4.27 ng/g. Contamination of aflatoxin in imported rice was dissimilar between different months. The highest levels of AFB 1 and AFT were detected in rice samples imported in September, while the lowest levels were in rice imported during November. Key words : Iran, aflatoxins, food safety, rice.

Occurrence of aflatoxins in milk thistle herbal supplements

Milk thistle (MT) dietary supplements are widely consumed due to their possible liver-health-promoting properties. As botanicals they can be contaminated with a variety of fungi and their secondary metabolites, mycotoxins. The aflatoxigenic fungus Aspergillus flavus has been previously isolated from these commodities. Currently, there is no published method for determining aflatoxins (AFs) in MT. Therefore, a liquid chromatography (LC) method validated for aflatoxin analysis in botanicals was evaluated and applied to MT. The method consisted of acetonitrile/water extraction, immunoaffinity column clean-up, LC separation, post-column photochemical reaction derivatisation and fluorescence detection. The average recoveries for AFs added to MT seeds, herb, oil-based liquid extract and alcohol-based liquid extract were 76% or higher. The mean relative standard deviation was <10%. The limit of detection (LOD) was 0.01 µg kg−1 and the limit of quantification (LOQ) was 0.03 µg kg–1. The method was used to conduct a small survey. A total of 83 MT samples from the US market were analysed. AFs were detected in 19% of the samples with levels ranging from 0.04 to 2.0 µg kg–1. Additionally, an aflatoxigenic A. flavus strain from ATTC and an A. parasiticus strain isolated from MT herb powder were found to produce high amounts of aflatoxins (11,200 and 49,100 µg kg–1, respectively) when cultured in MT seed powder. This is the first study reporting on aflatoxin contamination of MT botanical supplements and identifying methodology for AF analysis of these commodities.

Immersed sol‐gel based amino‐functionalized SPME fiber and HPLC combined with post‐column photochemically induced fluorimetry derivatization and fluorescence detection of pyrethroid insecticides from water samples

A method based on direct immersion solid-phase microextraction (DI-SPME) coupled with high performance liquid chromatography combined with post-column photochemically induced fluorimetry derivatization and fluorescence detection (HPLC-PIF-FD) was developed to extract three pyrethroid insecticides, i.e. cyfluthrin, cypermethrin, and flumethrin from water samples. A sol-gel based coating fiber using 3-(trimethoxysilyl propyl) amine as precursor was prepared and used for the extraction of the pyrethroids from groundwater samples. A post-column photochemical reactor was designed and constructed for the derivatization of these environmentally important pollutants to increase their fluorescence sensitivity and determination in HPLC. The parameters affecting extraction process (extraction time and temperature, pH, salt addition, and co-solvent) and desorption step (solvent, desorption time, and temperature) of the analytes from the sol-gel-based fiber, along with photochemical reaction conditions were investigated. The developed method proved to be relatively rapid, simple, and easy and offers high sensitivity and reproducibility. Linear dynamic ranges (LDR) for these insecticides were ranged between 0.25 to 50 microg/L. The regression coefficients were satisfactory (R(2) > 0.984) for these pyrethroids. The limits of detection and limits of quantification varied between 0.09 and 0.35 microg/L and 0.25 and 1.00 microg/L, respectively. Relative standard deviation RSDs values varied between 4.41% and 6.20%. Relative recoveries obtained from analysis of Jajroud river water sample ranged between 94% and 104%.

Selective determination method for measurement of nitrite and nitrate in water samples using high-performance liquid chromatography with post-column photochemical reaction and chemiluminescence detection

A simple, sensitive and selective method for the simultaneous determination of nitrite and nitrate in water samples has been developed. The method is based on ion-exchange separation, online photochemical reaction, and luminol chemiluminescence detection. The separation of nitrite and nitrate was achieved using an anion-exchange column with a 20 mM borate buffer (pH 10.0). After the separation, these ions were converted to peroxynitrite by online UV irradiation using a low-pressure mercury lamp and then mixed with a luminol solution prepared with carbonate buffer (pH 10.0). The calibration graphs of the nitrite and nitrate were linear in the range from 2.0 × 10 −9 to 2.5 × 10 −6 M and 2.0 × 10 −8 to 2.5 × 10 −5 M, respectively. Since the sensitivity of nitrite was about 10 times higher than that of nitrate, the simultaneous determination of nitrite and nitrate in the water samples could be efficiently achieved. This method was successfully applied to various water samples – river water, pond water, rain water, commercial mineral water, and tap water – with only filtration and dilution steps.

Determination of Thiamethoxam residues in honeybees by high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor

A method for the determination of Thiamethoxam in bee samples was set up by means of high performance liquid chromatography with an electrochemical detector and post-column photochemical reactor (HPLC–hν–ED). Analytical method was based on a rapid sample extraction procedure with acetone, followed by chromatographic separation into a C18-RP column isocratically operated by 60 mM phosphate buffer/acetonitrile (75/25) mobile phase at pH 2.7. A photochemical reactor was used as a tool to verify and eventually quantify the presence of Thiamethoxam in the samples by distinguishing it from interference contribution. Detection was performed with a potential of 880 mV after a photoactivation with a 254 nm light. The least detectable dose was 0.002 mg kg −1. Recovery rates ranged between 59.88 and 71.62%.

High-performance liquid chromatography of aromatic compounds with photochemical decomposition and tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection

A novel high-performance liquid chromatographic method for the determination of aromatic compounds based on the on-line photochemical degradation and subsequent tris(2,2′-bipyridine)ruthenium(III) chemiluminescence detection has been developed. Chemiluminescence intensity depended upon the number of aromatic rings, UV irradiation time, and variety of substituted functional groups. One of the decomposition products of aromatic compounds by UV irradiation was identified as oxalic acid. As one application of this methodology, determination of catechins in tea has been shown. Calibration graphs, based on standard (−)-epicatechin and (−)-epigallocatechin gallate solutions, were linear over the range of 0.1–50 μM. The detection limits (signal-to-noise ratio = 3) were 0.8 pmol for (−)-epigallocatechin gallate and 1.2 pmol for (−)-epicatechin. The high-performance liquid chromatography–chemiluminescence (HPLC–CL) detection method with a post-column photochemical reactor can be applied to the sensitive and selective determination of catechins in tea.

Determination of Imidacloprid and metabolites by liquid chromatography with an electrochemical detector and post column photochemical reactor

A procedure for the determination of Imidacloprid and its main metabolites was set up by means of liquid chromatography with an electrochemical detector and post-column photochemical reactor (LC- hν-ED). Sample clean-up was developed for bees, filter paper and maize leaves. Chromatographic conditions were based on a reversed-phase C-18 column operated by phosphate buffer 50 mM/CH 3CN (80/20, v/v) at pH 2.9. Detection of Imidacloprid and its metabolites was performed at a potential of 800 mV after photoactivation at 254 nm. Compared to conventional techniques such as gas chromatography/mass spectrometry (GC/MS) or LC coupled to other detectors, the present method allows simultaneous trace-level determination of both Imidacloprid (0.6 ng ml −1) and its main metabolites (2.4 ng ml −1).

High-performance liquid chromatographic assay of phosphate and organophosphorus pesticides using a post-column photochemical reaction and fluorimetric detection

A sensitive method for the post-column reaction detection of organophosphorus pesticides is described. The method relies on photolysis of the organocompounds by irradiation with a low-pressure mercury lamp (main spectral line, 254 nm) in the presence of peroxydisulfate. The resultant orthophosphate was reacted with molybdate to form molybdophosphoric acid, which subsequently reacted with thiamine to generate thiochrome. Finally, the fluorescence intensity of thiochrome was measured at 440 nm with excitation at 375 nm. Factors affecting the rate of these reactions were optimized so that its contribution to the total band-broadening was negligible. This detection system was used for the determination of phosphate, acephate and methamidophos, which were separated on an ODS column by isocratic reversed phase chromatography with acetonitrile–water as the mobile phase. A linear relationship between analyte concentration and peak area was obtained within the range 0.016–7.0 μg ml −1 with correlation coefficients greater than 0.9995 and detection limits between 4 and 12 ng ml −1. Intra- and inter-day precision values of about 1.2% R.S.D. ( n = 10) and 2.1% R.S.D. ( n = 30), respectively, were obtained. Pesticide residues below ng ml −1 levels could be determined in environmental waters when a preconcentration device was coupled on-line with the HPLC system. Detection limits as low as 0.01 ng ml −1 were achieved for only 250 ml of sample. In the analyses of vegetables and grains, the detection limit was about 1 μg kg −1.

光化学反応を利用したＨＰＬＣによる食品中の酢酸ｄｌ‐α‐トコフェロールおよびｄｌ‐α‐トコフェロールの定量

A reliable analytical method for the simultaneous determination of dl-alpha-tocopherol acetate and dl-alpha-tocopherol in foods was established by HPLC using post-column photochemical reaction with UV and fluorescence detection. For low-fat food such as fruit juice and vegetable sauce, the tocopherols were extracted with methanol containing 0.1% ascorbic acid and the extract solution was injected into the HPLC. For fatty foods such as butter and margarine, the tocopherols were extracted with a mixed solvent of acetonitrile-2-propanol (9:1) containing ascorbic acid. The extract was cleaned up using a Sep Pak plus C18 cartridge and the eluent from the cartridge was injected into the HPLC. The peaks corresponding to tocopherols on the chromatogram were confirmed by comparing their UV spectra with those of the standard mixture at lamp-on and lamp-off of the photochemical reactor. The recoveries of tocopherols from low-fat foods (orange juice and barbecue sauce) fortified at levels of 10 and 100 microg/kg each were 88.3 to 105.8% (RSD 0.5 to 6.0%) and those from the fatty foods (peanut butter and margarine) fortified at 100 microg/kg each were 57.1 to 88.3% (RSD 3.0 to 6.4%). The determination limits corresponded to 10 microg/kg of the tocopherols in the low-fat foods and 20 microg/kg in the fatty foods.

High-performance liquid chromatographic separation and quantification of citric, lactic, malic, oxalic and tartaric acids using a post-column photochemical reaction and chemiluminescence detection

An HPLC method was developed for the determination of citric, lactic, malic, oxalic and tartaric acids by chemiluminescent detection following online irradiation with visible light. The organic acids were irradiated with visible light in the presence of Fe 3+ and UO 2 2+ to generate Fe 2+, which was determined by measuring the chemiluminescence intensity in a luminol system in the absence of added oxidant. Factors affecting the photochemical and chemiluminescence reactions were optimised so that their contribution to the total band-broadening was negligible. The chromatographic separation was performed on a C 18 column under isocratic reversed-phase conditions using 0.005 M H 2SO 4 mobile phase. The optimised method was validated with respect to linearity, precision, limits of detection and quantification, accuracy specificity and robustness. The applicability of the assay was demonstrated by analysing these compounds in real samples such as milk, fruit juices, soft drinks, wine and beer.

Monitoring of phenylurea and propanil herbicides in river water by solid-phase-extraction high performance liquid chromatography with photoinduced-fluorimetric detection

The separation and determination of five herbicides, including propanil and the phenylureas diuron, isoproturon, linuron and neburon, has been performed by an HPLC method, using photochemically-induced fluorescence detection. The non-fluorescent herbicides were transformed into fluorescent compounds by post-column photochemical reaction. A 60:40 (v/v) acetonitrile–buffer solution of potassium phosphate dibasic (pH 7, 0.01 M) was used for the chromatographic elution to separate propanil, linuron and neburon. The overlapping of isoproturon and diuron peaks, in the selected conditions, was resolved by changing the initial movil phase composition to 50:50 (v/v) methanol–buffer solution of potassium phosphate dibasic (pH 7, 0.01 M). The procedure was applied with satisfactory results to the analysis of these herbicides in Guadiana river water samples (Badajoz, Spain), allowing the detection of herbicide residues in the order of μg l −1, by using a solid-phase extraction (SPE) pre-concentration step.