High Performance Liquid Chromatography Coupled With Fluorescence Detection Research Articles

Optimising urinary catecholamine metabolite diagnostics for neuroblastoma.

The analysis of urinary catecholamine metabolites is a cornerstone of neuroblastoma diagnostics. Currently, there is no consensus regarding the sampling method, and variable combinations of catecholamine metabolites are being used. We investigated if spot urine samples can be reliably used for analysis of a panel of catecholamine metabolites for the diagnosis of neuroblastoma. Twenty-four-hour urine or spot urine samples were collected from patients with and without neuroblastoma at diagnosis. Homovanillic acid (HVA), vanillylmandelic acid (VMA), dopamine, 3-methoxytyramine, norepinephrine, normetanephrine, epinephrine and metanephrine were measured by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) and/or ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry (UPLC-MS/MS). Catecholamine metabolite levels were measured in urine samples of 400 neuroblastoma patients (24-hour urine, n = 234; spot urine, n = 166) and 571 controls (all spot urine). Excretion levels of catecholamine metabolites and the diagnostic sensitivity for each metabolite were similar in 24-hour urine and spot urine samples (p > .08 and >.27 for all metabolites). The area under the receiver-operating-characteristic curve (AUC) of the panel containing all eight catecholamine metabolites was significantly higher compared to that of only HVA and VMA (AUC = 0.952 vs. 0.920, p = .02). No differences were observed in metabolite levels between the two analysis methods. Catecholamine metabolites in spot urine and 24-hour urine resulted in similar diagnostic sensitivities. The Catecholamine Working Group recommends the implementation of spot urine as standard of care. The panel of eight catecholamine metabolites has superior diagnostic accuracy over VMA and HVA.

OPTIMIZATION AND VALIDATION OF HPLC-FLD METHOD FOR DETERMINATION OF AFLATOXIN B1 AND PRODUCTION OF THIS MYCOTOXIN BY ASPERGILLUS SECTION FLAVI ISOLATES

A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) was evaluated for the monitoring of aflatoxin B1 (AFB1) production by Aspergillus section Flavi isolates, which were found in the commodities of Slovak origin (barley and tomatoes). Rapid validation of the method was performed and the method was applied for the determination of the mycotoxin. Based on the calculated validation characteristics, the method showed high sensitivity and reproducibility. The LOD and LOQ determined were 0.03 and 0.10 µg.mL-1 which is fully acceptable for the determination of the AFB1 in isolates from plant commodities. The calibration graph was linear in the concentration range of 0.01– 10.0 µg.mL-1. The optimized method was used for the analysis of the mycotoxin in three Aspergillus section Flavi isolates. The submerse cultivation was performed at 25±1°C in yeast extract sucrose liquid medium and material was collected on the 7th, 14th and 21st day of cultivation. Simple sample extraction was carried out with use of ethyl acetate, and measurement was done with use of the mobile phase consisting of water and methanol by HPLC-FLD. Production of the AFB1 by isolates was ranging from <LOD (0.03 µg. mL-1) to 49.38 µg.mL-1 with the highest concentration on the 14th day of cultivation. The maximum level of AFB1 was achieved by the Isolate 1 (isolated from barley, Kolíňany, 2018).

Simultaneous Detection of Ochratoxin A and Aflatoxins in Industrial and Traditional Red and isot Pepper Flakes along with Dietary Exposure Risk Assessment

This study focused on the co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in traditionally and industrially dried red pepper flakes (DRPFs) and isot pepper flakes (IPFs). Following the multitoxin immunoaffinity column (IAC) clean-up, high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) was used to quantify AFs and OTA. The limit of detection (LOD) and limit of quantification (LOQ) values were 0.11 and 0.18 μg kg–1 (AFB1), 0.04 and 0.08 μg kg–1 (AFB2), 0.13 and 0.18 μg kg–1 (AFG1), 0.04 and 0.11 μg kg–1 (AFG2), and 0.10 and 0.21 μg kg–1 (OTA), respectively. AFB1, AFB2, AFG1, and OTA were found to be positive in 93, 74, 17, and 94% of all samples, respectively. The contamination levels in positive samples ranged from 0.23 to 38.69, 0.04 to 2.14, 0.13 to 0.88, and 0.18 to 52.19 μg kg–1 for AFB1, AFB2, AFG1, and OTA, respectively, while no AFG2 was found above the detection limit (0.04 μg kg–1). None of the industrial isot peppers exceeded the European Union limits, while the levels of AFB1 (5 μg kg–1), total AFs (10 μg kg–1), and OTA (20 μg kg–1) of the traditional peppers were above the limit by 30% (16/54), 26% (14/54), and 4% (2/54), respectively. Co-occurrence of AFB1-AFB2-OTA was the most frequent, accounting for 54% (29/54) of all samples. At the upper bound (UB), estimated average exposure to AFB1, total AFs, and OTA was determined to be 0.175, 0.189, and 0.124 ng kg–1 bw day–1 in all samples, respectively. The margin of exposure (MoE) value of AFB1 and total AFs was found to be 977 and 909, indicating high health concerns compared to OTA (MoE >10,000). AFB1 and total AFs may result in 0.0058 and 0.0062 liver cancer cases/100,000 person/year at UB, respectively, and weekly OTA exposure was 0.868 ng kg–1 bw, well below the provisional tolerable weekly intake, hence not of health concern. AFs exposure could endanger health, whereas OTA posed no toxicological concerns through dried red pepper consumption

Alginate oligosaccharides enhance diffusion and activity of colistin in a mucin-rich environment

In a number of chronic respiratory diseases e.g. cystic fibrosis (CF) and chronic obstructive pulmonary disease (COPD), the production of viscous mucin reduces pulmonary function and represents an effective barrier to diffusion of inhaled therapies e.g. antibiotics. Here, a 2-compartment Transwell model was developed to study impaired diffusion of the antibiotic colistin across an artificial sputum (AS) matrix/medium and to quantify its antimicrobial activity against Pseudomonas aeruginosa NH57388A biofilms (alone and in combination with mucolytic therapy). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) revealed that the presence of AS medium significantly reduced the rate of colistin diffusion (> 85% at 48 h; p < 0.05). Addition of alginate oligosaccharide (OligoG CF-5/20) significantly improved colistin diffusion by 3.7 times through mucin-rich AS medium (at 48 h; p < 0.05). Increased diffusion of colistin with OligoG CF-5/20 was shown (using confocal laser scanning microscopy and COMSTAT image analysis) to be associated with significantly increased bacterial killing (p < 0.05). These data support the use of this model to study drug and small molecule delivery across clinically-relevant diffusion barriers. The findings indicate the significant loss of colistin and reduced effectiveness that occurs with mucin binding, and support the use of mucolytics to improve antimicrobial efficacy and lower antibiotic exposure.

A simple and low-cost sample preparation for the effective extraction, purification and enrichment of aflatoxins in wheat by combining with ionic liquid-based dispersive liquid–liquid microextraction

The direct extraction, purification, and enrichment of four aflatoxins (AFs) in wheat were achieved by combining solvent extraction and ionic liquid-based dispersive liquid–liquid microextraction (IL-DLLME). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) was applied for the determination of AFs. The feature of the developed technique is that some tedious and time-consuming steps are avoided and the organic solvent consumption is low. The IL-DLLME allows the isolation, purification and enrichment of AFs from the methanol extract using a simple procedure. The effective enrichment was mainly due to the multiple-interaction, including hydrophobic, π-π and hydrogen bonding interactions, between IL and AFs. The parameters affecting the extraction efficiency were thoroughly investigated. This extraction method permits the determination of four AFs with LOD (S/N = 3) and LOQ (S/N = 10) in the range of 0.015–0.20 μg kg−1 and 0.045–0.75 μg kg−1, respectively. The recoveries from four spiked wheat samples ranged from 80.8% to 112.5% with RSDs of 0.8%-4.7% (n = 3), demonstrating the great potential of this proposed procedure for the analysis of AFs in solid samples.

Integrative strategy to determine residual proteins in cefaclor produced by immobilized penicillin G acylase

There is a growing trend in the pharmaceutical industry towards substituting conventional chemical synthesis routes of semi-synthetic β-lactam antibiotics (SSBAs) through environmentally sustainable enzymatic processes. These have advantages such as cost reduction in terms of solvent and waste treatment and time saving owing to fewer reaction steps. Penicillin G acylase (PGA) is an industrially important enzyme that is mainly used to catalyze the synthesis of SSBAs. In this study, we established an integrative strategy using three different analytical methods for determining the PGA-associated residual protein content, which is a critical quality issue in the end product. Cefaclor was taken as representative example of SSBAs. High-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) allowed the routine analysis of PGA residual proteins and other low molecular weight (MW) impurities with high detection specificity and sensitivity, comparable to those of the Bradford assay and microfluidic protein chip electrophoresis. However, these latter two methods were superior for quantitative and qualitative analysis, respectively, and should be regarded as necessary adjuncts to the HPLC-FD method. By combining the three methods, trace levels of residual proteins were detected in four (out of 13) cefaclor bulk samples from two different manufacturers, with a major protein MW of ∼63 kDa. This suggests that the higher MW PGA subunit tends to persist in the end product. The integrative determination strategy described here can be used to evaluate SSBA bulk samples and monitor the process of SSBA manufacturing by enzymatic methods, especially in terms of inter-batch consistency and process stability.

Aflatoxins and ochratoxin A in chocolate products in Turkey

ABSTRACTThis survey describes the occurrence and levels of AFs and OTA in chocolate products consumed in Turkey. A total of 130 samples, including bitter chocolate, milk chocolate and chocolate wafers were analysed for these mycotoxins by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). The values of recovery (81–92%) and precision (RSD < 9%) fulfilled the requirements of EC Regulation No. 401/2006. OTA was the most prevalent mycotoxin, with an incidence of 46.7% in bitter chocolate, 22.8% in milk chocolate and 17.4% in chocolate wafers, ranging from 0.18 to 0.75 μg kg−1. AFs were detected in 13.3% of bitter chocolate, in 19.6% of milk chocolate and in 8.7% of chocolate wafers, in concentrations ranging from 0.15 to 2.04 μg kg−1.

Magnetic nanocomposite as sorbent for magnetic solid phase extraction coupled with high performance liquid chromatography for determination of polycyclic aromatic hydrocarbons

Magnetic composites-based adsorbents have received considerable attention in recent years due to their inherent superparamagnetic properties making them desirable for magnetic field assisted separation and enrichment. In this work, a novel magnetic adsorbent, Fe3O4@SiO2@MMTA-Au, was synthesized via 2-mercapto-4-methyl-5-thiazoleacetic acid bonding Au (MMTA-Au) deposited on Fe3O4@SiO2. The as synthesized core shell structured adsorbent, Fe3O4@SiO2@MMTA-Au magnetic nanocomposite was characterized with transmission electron microscopy (TEM), vibrating sample magnetometer (VSM), Fourier transform-infrared (FT-IR) spectroscopy, X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS). This material was used as adsorbent of magnetic solid phase extraction (MSPE) to extract 14 polycyclic aromatic hydrocarbons (PAHs) of interest from water samples followed by the determination of their concentrations with high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). The effects of four parameters of the MSPE were investigated, including the adsorbent mass, extraction time, extraction solvent and adsorption time. Under the optimized conditions, the enrichment factors (EFs) achieved were in the range of 80–98 and the detection limits were from 0.25 to 37.5 ng/L together with the good recoveries ranging from 87.8 to 120.0% of PAHs from water spiked. The developed method was successfully applied to the analysis of PAHs in water samples collected from river and rainfall.

Aflatoxin B1, ochratoxin A and zearalenone in sorghum grains marketed in Tunisia

ABSTRACTA total of 64 samples of sorghum (37 Tunisian sorghum samples and 27 Egyptian sorghum samples) were collected during 2011–2012 from markets in Tunisia. Samples were analysed for contamination with aflatoxin B1, ochratoxin A and zearalenone by High-Performance Liquid Chromatography Coupled with Fluorescence Detection (HPLC-FLD). Aflatoxin B1 was found in 38 samples in the range 0.03–31.7 µg kg−1. Ochratoxin A was detected in 24 samples with concentrations ranging from 1.04 to 27.8 µg kg−1. Zearalenone was detected in 21 samples and the concentration varied between 3.7 and 64.5 µg kg−1. ANOVA analysis of the influence of the country of origin on the incidence and concentration of mycotoxins in the samples studied showed no significant difference (P > 0.05) between the two batches of samples for each of the three mycotoxins studied. The studied mycotoxins contaminate sorghum and may also co-exist because of the diversity of the mycobiota in this cereal.

Mold infestation and aflatoxins production in traditionally processed spices and aromatic herbs powder mostly used in West Africa.

Mold infestation and occurrence of aflatoxins were investigated in 66 samples of dried spices and aromatic herbs powder (SAH) as commercialized in Benin and its neighboring countries. The samples were randomly collected from markets, supermarkets, and processing sites. Mold counts were enumerated according to standard method and aflatoxins levels were assessed using high‐performance liquid chromatography coupled with fluorescence detection (HPLC‐FLD). The results revealed that mold counts of samples ranged between 2.62 and 4.34 LogCFU/g. Aflatoxin B1 contents were between 0.46 μg/kg and 84.84 μg/kg with 40% of samples exceeding the recommended limit of 5 μg/kg. Aflatoxins G1 and G2 levels were low in general with means values varying from 0.24 to 8.56 μg/kg, and 0.11 to 3.68 μg/kg, respectively. Fifty‐two percent (52%) of samples analyzed contained total aflatoxins levels lower than the stipulated limit of 10 μg/kg, whereas 92% of them were contaminated at various levels with one type aflatoxin, B1 or B2, G1 or G2. This study provides the first information about the occurrence of aflatoxins in the common spices used in West Africa.

Aflatoxins in hazelnuts and dried figs: Occurrence and exposure assessment

A total of 300 samples of hazelnuts and dried fig were analysed for the incidence of any aflatoxins (AFs). High-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method was used to quantify the amounts of AFs. The limit of quantification varied from 0.21 to 0.30μgkg−1. No AFs were detected in shells of the hazelnuts, while six raw hazelnut kernel samples (12%) and five roasted hazelnut kernel samples (8.3%) contained AFs ranging from 0.09 to 11.3μgkg−1 and from 0.17 to 11.2μgkg−1, respectively. Sixteen dried fig samples (12.3%) contained AFs ranging from 0.1 to 28.2μgkg−1 and a mean value of 3.8μgkg−1. Three hazelnuts and six dried fig samples exceeded the European maximum limits (MLs) of 5 and 2μgkg−1 for aflatoxin B1 (AFB1), respectively. The contribution of hazelnuts to AFs exposure is higher than that of dried figs.

Aflatoxins in dairy cow feed, raw milk and milk products from Turkey

ABSTRACTThis study aims to detect aflatoxins (AFs) in dairy cow feed, milk and milk products using a high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method. All the validation parameters met the method performance criteria of the European Union. The samples comprised 76 dairy cow feeds and 205 milk and milk products (including yoghurt and yoghurt-based beverage, ayran). AFs were present in 26.3% of the feed samples. Two feed samples exceeded the maximum limit (ML) of 5 µg kg−1 for AFB1 as established by the EU. Nineteen milk samples (21.1%) contained aflatoxin M1 (AFM1) of which three exceeded the EU ML of 0.05 µg l−1. In addition, only two yoghurt samples and one ayran sample contained AFM1, but the levels were lower than the EU ML.

First report: Exposure estimates to ochratoxin A through wheat bread and rice intake in Turkey

A total of 160 samples of fresh wheat bread and rice from Turkey were analysed for the presence of ochratoxin A (OTA). The samples were analysed using a high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). The method was previously validated and showed good selectivity, linearity, sensitivity and accuracy. The uncertainty associated with the analytical method was less than 20%. OTA was found in 9.8% of wheat bread (10 out of 102 samples) and 5.2% of rice (3 out of 58 samples) at levels ranging from <LOQ to 2.83 μg/kg, and from <LOQ to 0.98 μg/kg, respectively. Neither wheat bread nor rice samples contained OTA at levels higher than EU maximum limit (ML) of 3 μg/kg. The estimated daily intakes of OTA through the consumption of wheat bread and rice were 0.85 and 0.02 ng/kg b.w./day for general Turkish consumers, respectively. To our knowledge, this is the first report on exposure to OTA through the consumption of wheat bread and rice for Turkish consumers.

Contamination of common spices by aflatoxigenic fungi and aflatoxin B1in Algeria

Spices are usually produced in areas where the climatic conditions are favourable to growth of toxigenic fungi and production of mycotoxins. This study assesses the occurrence of aflatoxigenic fungi and aflatoxin B1 (AFB1) in spices marketed in Algeria. A total of 44 spice samples (4 for each type of spice) composed of aniseed, black pepper, caraway, cinnamon, coriander, cumin, ginger, red pepper, saffron, sweet cumin, and sweet pepper were collected from four popular markets located in Algeria. Mycological analysis of the spice was by dilution plating while AFB1 contamination levels were determined by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) after post-column derivatisation. The commonly isolated fungi were species of Aspergillus (56.4%), Penicillium (25.1%), Mucor (12.8%) and Eurotium (5.7%). Species belonging to Aspergillus section Flavi represented 28.9% of the total Aspergilli. The aflatoxin producing ability of isolates belonging to Aspergillus section Flavi was determined on coconut agar medium and confirmed by thin layer chromatography and HPLC-FLD. Ninety-four isolates (38.4%) of the 245 Aspergillus section Flavi examined produced aflatoxins. The most frequent chemotypes (84%) correspond to isolates able to produce both aflatoxin B and cyclopiazonic acid followed by the producers of only aflatoxin B. Twenty-three (63.9%) of the 36 spices contained AFB1 at levels ranging from 0.10 to 26.50 μg/kg. Two saffron (24.34 and 26.50 μg/kg) and two sweet cumin (14.65 and 19.07 μg/kg) samples were above the Algerian regulatory limit of 10 μg/kg. This work represents the first report about the occurrence of aflatoxigenic fungi and AFB1 in the common spices in Algeria.

Determination of aflatoxins in walnut sujuk and Turkish delight by HPLC-FLD method

This study aims to assess the risk of aflatoxins (AFs) in traditional confectionery products (walnut sujuk and Turkish delight) of Turkey. A high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method was used for the determination of AFs. Evaluation of the method showed good selectivity, linearity, recovery and precision. The limit of quantification (LOQ) ranged from 0.106 to 0.374 μg kg−1. The expanded measurement uncertainty was less than 40% for all target analytes. The validated method was successfully applied to the determination of AFs in 112 traditional confectionery products containing nuts (hazelnuts and walnuts). AFs were detected in 43.8% of walnuts and 60.9% of hazelnuts used as ingredients in walnut sujuk and Turkish delight and at levels ranging from 0.58 to 15.2 μg kg−1 and 0.43–63.4 μg kg−1, respectively. This means that AFs levels in walnut sujuk and Turkish delight were up to levels of 6.1 and 9.5 μg kg−1, respectively. Six walnut samples and twenty-one hazelnut samples were above the EU maximum limits (MLs) of 2 and 5 μg kg−1 for aflatoxin B1 (AFB1), respectively.

Determination of 3-OHB[a]P and (+)-anti-BPDE in Rats Blood and Brain Tissue of B[a]P Exposure by HPLC with Fluorescence Detection

The aim of this paper was to develop an analytical method for determining the levels of the Benzo[a]pyrene (B[a]P) metabolites, 3-hydroxybenzo(a)pyrene (3-OHB[a]P), and (+)-anti-benzo(a)pyrene diol-epoxide( BPDE, combined with DNA to formate adducts) in the blood and brain tissue of B[a]P-exposed rats using high-performance liquid chromatography coupled with fluorescence detection (HPLC/FD). The chromatographic separation was performed with a CenturySIL BDS C18 column (150 mm × 4.6 mm, 5 μm) using mobile phases consisted of different methanol–water ratios [97:3 or 55:45 (v/v)] and different pHs (4.5 or 7.0). The amounts of 3-OHB[a]P and B[a]P-tetrols I-1 released after acid hydrolysis of (+)-anti-BPDE were detected by fluorescence at λ ex/λ em = 365/450 nm and λ ex/λ em = 245/395 nm. The calibration curves were linear (r 2 > 0.9990) for 3-OHB[a]P within 27–4320 ng mL−1 and for (+)-anti-BPDE within 1–160 ng mL−1, and the limits of detection (S/N = 3) of 3-OHB[a]P and (+)-anti-BPDE were 0.2 and 0.3 ng mL−1, respectively. Variation in intra- and inter-day assay as well as the data concerned with the stability of these compounds indicated no significant degradation under current experimental conditions. The average recoveries of the compound were in the range of 73.6 ± 5.0–113.5 ± 6.7 % for 3-OHB[a]P and 79.3 ± 6.2–141.2 ± 13.8 % for (+)-anti-BPDE. Our study have provided a sensitive, reliable, and rapid HPLC/FD-based method for the analysis of 3-OHB[a]P and (+)-anti-BPDE in rats blood and brain tissue.

Development of a sensitive and reliable high performance liquid chromatography method with fluorescence detection for high-throughput analysis of multi-class mycotoxins in Coix seed

As an edible and medicinal plant, Coix seed is readily contaminated by more than one group of mycotoxins resulting in potential risk to human health. A reliable and sensitive method has been developed to determine seven mycotoxins (aflatoxins B1, B2, G1, G2, zearalenone, α-zearalenol, and β-zearalenol) simultaneously in 10 batches of Coix seed marketed in China. The method is based on a rapid ultrasound-assisted solid–liquid extraction (USLE) using methanol/water (80/20) followed by immunoaffinity column (IAC) clean-up, on-line photochemical derivatization (PCD), and high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD). Careful optimization of extraction, clean-up, separation and detection conditions was accomplished to increase sample throughput and to attain rapid separation and sensitive detection. Method validation was performed by analyzing samples spiked at three different concentrations for the seven mycotoxins. Recoveries were from 73.5% to 107.3%, with relative standard deviations (RSDs) lower than 7.7%. The intra- and inter-day precisions, expressed as RSDs, were lower than 4% for all studied analytes. Limits of detection and quantification ranged from 0.01 to 50.2μgkg−1, and from 0.04 to 125.5μgkg−1, respectively, which were below the tolerance levels for mycotoxins set by the European Union. Samples that tested positive were further analyzed by HPLC tandem electrospray ionization mass spectrometry for confirmatory purposes. This is the first application of USLE-IAC-HPLC-PCD-FLD for detecting the occurrence of multi-class mycotoxins in Coix seed.

Degradation products of proguanil — 4-chloroaniline and related components with regard to genotoxicity

AbstractIn the event of genotoxic impurities in drug products, knowledge of the origin and fate of these components is of particular importance. In the present study, commercially available proguanil tablets (Paludrine®) were investigated in respect of formation of the main degradation product, 4-chloroaniline (PCA), which is known to be genotoxic. The investigations made use of high performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) and gas chromatography coupled with flame ionisation detection (GC-FID) systems. In addition, proposals for the structure of further proguanil degradation products were developed based on results obtained by mass spectroscopy (HPLC-MS). Database analysis using DEREK, MCASE, and Vitic was performed to obtain an initial toxicological evaluation of the proposed chemical structures. Finally, the absence of the newly established structures in stored proguanil tablets was verified.

Natural co-occurrence of aflatoxins and ochratoxin A in spices

The present study aims to determine the co-occurrence of aflatoxins (AFs) and ochratoxin A (OTA) in spices marketed in Turkey. During the period from November 2010 to August 2011, 105 samples of spices were checked for targeted mycotoxins. The mycotoxins were determined by high-performance liquid chromatography coupled with fluorescence detection (HPLC-FD) after immunoaffinity column (IAC) clean-up. The method was validated for selectivity, sensitivity, accuracy and repeatability as relative standard deviation (RSD). Good recoveries (80.7–95.7%) were obtained for mycotoxins in each food matrix, with RSD values lower than 15%.Aflatoxins were detected in 79.2% of red chilli flake, 63.6% of red chilli powder, 30.4% of black pepper powder and 21.1% of cumin samples, while none of the cinnamon powder samples contained AFs at detectable levels. Four red chilli flake and three red chilli powder samples were above the EU regulatory limit of 5μgkg−1 for AFB1. OTA was found in 75% of red chilli flake, 54.5% of red chilli powder, 17.4% of black pepper powder, with four red chilli flake and three red chilli powder samples exceeding EU limit of 30μgkg−1. No OTA was found in cinnamon powder samples, while detectable levels of OTA were found in only one cumin sample. The co-occurrence of AFB1 and OTA was detected in 62.5% of red chilli flake, 40.9% of red chilli powder and 4.3% of black pepper powder samples. This study reports for the first time the natural co-occurrence of AFs and OTA in spices from Turkey.

Passage of 4-nonylphenol across the human placenta

4-nonylphenol (4-NP) is an estrogenic endocrine active chemical that is present in detergents and is known to contaminate food and drinking water. The aim of the present study was to evaluate whether 4-NP crosses the human placenta and if so, to what extent. Human placentae obtained from healthy term singleton pregnancies were utilized in a dual ex-vivo re-circulating model of placental perfusion. Six placentae were perfused for 180 min following addition of 4-NP (30 ng/mL) to the maternal perfusate. Antipyrine and FITC dextran were used as positive and negative controls respectively to validate the integrity of the circuits. 4-NP was measured by High Performance Liquid Chromatography coupled with Fluorescence Detection (HPLC-FD). After 180 min of perfusion the transfer percentages for antipyrine and 4-NP were 25.6 ± 1.4% (mean ± s.e.m, n = 6) and 22.75 ± 3.76% respectively and the transfer index for 4-NP was 0.8. We conclude that the intact form of 4-NP at environmentally relevant concentrations can transfer across the human placenta albeit at a slow rate.