High-performance Liquid Chromatography With Fluorescence Detection Research Articles

Surveillance of aflatoxin content in dairy cow feedstuff from Navarra (Spain)

Aflatoxins (B1, B2, G1 and G2) are produced by the fungi Aspergillus (A. flavus and A. parasiticus) in substrates used in cattle feed manufacturing. Aflatoxin M1 (AFM1) is a major metabolite of Aflatoxin B1 (AFB1) which may be present in milk from animals that consume contaminated feed. Levels of aflatoxins in 78 dairy cow feedstuff samples from 40 farms located in Navarra were determined by HPLC-FLD (High Performance Liquid Chromatography with fluorescence detection) and post-column derivatization. The influence of geographical location, season and type of feeding system on aflatoxin content was studied. The climatic profile of AFB1 pointed to spring as the season with the highest aflatoxin level (0.086μg/kg), followed by winter and summer (0.075 and 0.030μg/kg, respectively), and to a lesser degree, autumn (0.017μg/kg). Moreover, wet and dry TMR (Total Mixed Ration) feeding systems (i.e. AFB1: 0.076 and 0.068μg/kg; Aflatoxin G1 (AFG1): 0.050 and 0.011μg/kg, respectively) showed a greater content of the analyzed aflatoxins in comparison with compound feed (i.e. AFB1: 0.039μg/kg; AFG1: 0.007μg/kg). The fact that the majority of the samples collected were based on compound feed shows that this type was preferred by most dairy farmers. The undetectable levels of aflatoxins in the organic homemade compound feedstuff are also worth mentioning. While none of the feedstuff samples contained amounts over those permitted under European legislation (5μg/kg), the theoretical extrapolation of the carryover rate suggested in previously published experiments of AFB1 to AFM1 in secreted cow's milk predicts that only one of the feed samples studied had a positive aflatoxin level (53.4ng/kg) higher than the legal limit for raw cow's milk.

Validation of a method of high performance liquid chromatography with fluorescence detection for melamine determination in UHT whole bovine milk

A method of high performance liquid chromatography with fluorescence detection (HPLC-Fluo), based on previous studies of the natural fluorescence emission of melamine (250/365 nm), was developed and validated for melamine determination as an adulterant of bovine UHT whole milk. The clean up treatment of milk samples by deproteinization was thoroughly optimized. No matrix effect was observed and a linear range from 0.05 to 10.0 μg mL−1 was obtained, with a value of R2 equal to 0.9998. The limits of detection and quantification of melamine were 0.0081 and 0.027 μg mL−1 respectively, or 0.023 and 0.076 μg g−1 of milk, respectively, considering the sample dilution during extraction. Extracts of milk samples fortified with melamine at three concentration levels, two of which corresponded to the levels established by WHO for melamine in foods, led to an overall mean recovery of 95.4 ± 1.2% (n = 9). This recovery value satisfies the performance criteria established by the Codex Alimentarius for analytical methods suitable for determination of food residues, demonstrating the usefulness and effectiveness of the proposed method.

Natural incidence of Fusarium species and fumonisins B1 and B2 associated with maize kernels from nine provinces in China in 2012

Fusarium species, which can produce mycotoxins, are the predominant pathogens causing maize ear rot, a disease that results in severe economic losses and serves as a potential health risk for humans and animals. A survey was conducted in 2012 to investigate the contamination of maize by Fusarium species and fumonisins B1 and B2. A total of 250 maize samples were randomly collected from nine provinces (Hebei, Shanxi, Inner Mongolia, Yunnan, Sichuan, Guizhou, Heilongjiang, Liaoning and Ningxia) in China. Fusarium species were isolated and identified using morphological (electron microscope) and molecular methods (polymerase chain reaction (PCR) and sequencing). Fumonisins B1 and B2 were analysed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD) with OPA (2-Mercaptoethanol, o-phthaldialdehyde) post-column derivatisation. A total of 2321 Fusarium isolates (20.7%) were obtained from all the samples. These isolates included nine Fusarium species, namely, F. graminearum, F. verticillioides, F. subglutinans, F. proliferatum, F. temperatum, F. oxysporum, F. equiseti, F. meridionale and F. chlamydosporum. The incidence of occurrence of Fusarium species in Guizhou was the highest, while in Inner Mongolia it was the lowest. F. verticillioides was the dominant species of maize ear rot in Liaoning, Sichuan, Hebei and Ningxia. F. graminearum was the dominant species in Yunnan, Guizhou and Shanxi. F. subglutinans was the dominant species in Heilongjiang. F. verticillioides and F. graminearum percentages were the same in Inner Mongolia. The incidence of fumonisins in Liaoning was high (up to 81.0%) and in Heilongjiang low (up to 10.3%). Except Shanxi, more than 50% of maize samples from other provinces were contaminated with fumonisins, with concentrations less than 500 ng g−1. About 33% of maize samples from Yunnan were contaminated with high levels of fumonisins, and average of fumonisin levels were 5191 ng g−1. Fusarium species causing maize ear rot in different areas in China were highly diverse and such areas with exposure to high levels of fumonisin contamination have a potential health risk for human and animals.

In-house validation of a method for determining aflatoxins B1, B2, G1 and G2 in wheat and wheat by-products

Aflatoxins are naturally occurring carcinogenic substances, extremely toxic to humans, which have been identified in wheat and wheat by-products. The use of reliable analytical methods to evaluate and monitor such contaminants is extremely important. This study aimed to in-house validate a methodology to determine aflatoxins B1, B2, G1 and G2 in wheat grains using pre-column derivatization and quantification by High Performance Liquid Chromatography with fluorescence detection (HPLC-FLD). Three methods were evaluated and the most suitable one was the method based on the extraction with chloroform, removal of interfering chemicals by filtration, liquid-liquid partition with hexane-methanol-water and methanol-water-chloroform and pre-column derivatization with trifluoroacetic acid. The method showed a Relative Standard Deviation lower than 15% and recovery values in the 70-110% range, with limits of detection and quantification (0.6 µg kg-1 and 1.2 µg kg-1, respectively) below the maximum level of aflatoxins allowed in wheat and wheat by-products by the European Commission (4.0 µg kg-1) and by the Brazilian legislation (5.0 µg kg-1). Using the validated method, aflatoxins were quantified in 20 commercial samples of wheat grains, wheat bran, whole wheat flour and refined wheat flour intended for direct human consumption. Six samples (30%) were positive for aflatoxins and all samples presented levels below the maximum limit stipulated by the Brazilian legislation.

Systematic optimization of an SPE with HPLC-FLD method for fluoroquinolone detection in wastewater

This paper describes a selective and ultra-sensitive analytical method for simultaneous determination of 11 fluoroquinolone (FQ) antibiotics in environmental and wastewater samples. The method employs offline solid-phase extraction (SPE) and reversed-phase high performance liquid chromatography with fluorescence detection (HPLC-FLD). A weak cation exchange SPE protocol was developed with a novel loading volume optimization algorithm and a methanol cleanup step to remove background organic matter. Various parameters were optimized to recover FQs from water/wastewater and analyte recovery was generally greater than 80%. Chromatographic separation of the 11 FQs was achieved on a 150mm pentafluorophenyl column using a gradient elution scheme with methanol, acetonitrile, and 20mM phosphate buffer (pH=2.4). Excitation and emission wavelengths were individually optimized for each FQ using fluorescence spectroscopy; the excitation and emission wavelengths were 276–296nm and 444–506nm, respectively. Instrumental quantitation limits were 20–100pg of mass injected. Of the 11 FQs investigated, seven (i.e., ciprofloxacin, difloxacin, enrofloxacin, fleroxacin, norfloxacin, moxifloxacin, and ofloxacin) were detected during a four-month sampling campaign of wastewater and wastewater-impacted surface water. Concentrations of FQs in raw wastewater, wastewater effluent, and wastewater-impacted surface water were 5–1292, 2–504, and 4–187ng/L, respectively.

Molecularly imprinted polymers for cleanup and selective extraction of curcuminoids in medicinal herbal extracts.

This paper describes the synthesis of novel molecularly imprinted polymers (MIPs), prepared by a noncovalent imprinting approach, for cleanup and preconcentration of curcumin (CUR) and bisdemethoxycurcumin (BDMC) from medicinal herbal extracts and further analysis by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Two molecular mimics, a mixture of reduced BDMCs and 4-(4-hydroxyphenyl)-2-butanone (HPB), have been synthesized and applied as templates for MIP synthesis. The polymers were prepared using N-(2-aminoethyl) methacrylamide (EAMA) as functional monomer, ethylene glycol dimethacrylate (EDMA) as the cross-linker (in a 1:5 molar ratio), and a mixture of acetonitrile/dimethylsulfoxide (90%, v/v) as porogen. MIPs prepared using a mixture of reduced BDMCs as template showed higher selectivity for CUR and BDMC than those obtained with HPB, with imprinting factors of 3.5 and 2.7 for CUR and BDMC, respectively, using H2O/acetonitrile (65:35, v/v) as mobile phase. The adsorption isotherms for CUR in the MIP and the nonimprinted polymer (NIP) were fitted to the Freundlich isotherm model, and the calculated average binding affinities for CUR were (17 ± 2) and (8 ± 1) mM(-1) for the MIP and the NIP, respectively. The polymers were packed into solid-phase extraction (SPE) cartridges, and the optimized molecularly imprinted solid-phase extraction (MISPE-HPLC) with fluorescence detection (FLD) method allowed the extraction of both curcuminoids from aqueous samples (50 mM NH4Ac, pH 8.8) followed by a selective washing with acetonitrile/NH4Ac, 50 mM at pH 8.8 (30:70%, v/v), and elution with 3 × 1 mL of MeOH. Good recoveries and precision ranging between 87 and 92%, with relative standard deviation (RSD) of <5.3% (n = 3), were obtained after the preconcentration of 10-mL solutions containing both CUR and BDMC at concentrations in the range of 0-500 μg L(-1). The optimized method has been applied to the analysis of both curcuminoids in medicinal herbal extracts.

Efficient analysis of selected estrogens using fabric phase sorptive extraction and high performance liquid chromatography-fluorescence detection

A simple, fast and sensitive analytical method using fabric phase sorptive extraction (FPSE) followed by high performance liquid chromatography with fluorescence detection (HPLC-FLD) has been developed for efficient quantification of biologically important molecules e.g., 17α-ethynylestradiol (EE2), β-estradiol (E2) and bisphenol A (BPA). FPSE is a new sorptive extraction technique that integrates the advantages of permeable sol–gel derived hybrid organic–inorganic sorbents with flexible and permeable fabric substrates, resulting in a highly efficient and sensitive extraction media that can be introduced directly into any kind of fluidic matrix. Various factors affecting the performance of FPSE technique were optimized. The chromatographic separation was carried using mobile phase acetonitrile/methanol/water (30:15:55; v/v) at a flow rate 1.0mL/min on C18 column with fluorescence detection (λex=280nm and λem=310nm). The calibration curves of the target analytes were prepared with good correlation coefficient values (R2>0.992). Limit of detection (LOD) values range from 20 to 42pg/mL. The developed method was applied successfully for the analysis of estrogen molecules in urine and various kinds of aqueous samples.

Identification of ochratoxin A in Chinese spices using HPLC fluorescent detectors with immunoaffinity column cleanup

Spices can be heavily contaminated with Penicillium verrucosum and Aspergillus ochraceus, which develop during cultivation and after harvest if relative humidity is not controlled during storage. The present study was undertaken with the aim to provide an analytical method to detect OTA in spices commercialized in China, specifically to develop a simple and rapid method for the determination of Ochratoxin A content in spices via immunoaffinity column and high performance liquid chromatography with fluorescence detection (HPLC-FLD). The recovery (75.0–102.0%) and repeatability (RSD < 12) are acceptable according to the requirements of the Commission Regulation (EC) No. 401/2006. The method is accurate, sensitive and rapid. A total of 480 spice products were analyzed with the amended analytical methods used in our study, which involved the extraction of OTA, immunoaffinity cleanup and high performance liquid chromatography determination with fluorescence detection. This report is the first describing the OTA presence in retail spices (pepper, chili, prickly ash, cinnamon, aniseed, fennel, curry powder and cumin) in China. The percentage of OTA contamination in spice product is 9.6%, and two chili products were above the levels permitted by the EU for safe consumption. Surveillance of a large number of suspected spice products should be continuous and widespread.

Validated RP-HPLC Method for Simultaneous Determination of Tocopherols and Tocotrienols in Whole Grain Barley Using Matrix Solid-Phase Dispersion

The vitamers of vitamin E such as α-, β-, γ-, and δ-tocotrienol and α-, β-, γ-, and δ- tocopherol are important phytochemical compounds with antioxidant activity and with potential benefits for human health. A high-performance liquid chromatography with fluorescence detection (HPLC-FLD) method was validated for their determination in whole grain barley samples. Tocol extraction was performed by an optimized matrix solid-phase dispersion (MSPD) protocol with neutral alumina (0.5 g) as the dispersion agent and methanol (5 mL) as the elution solvent. The analytical column was an Eclipse XDB C18 column (150 × 4.6 mm, 5 μm) and it was operated at room temperature. Mobile phase was consisted of methanol/acetonitrile/i-propanol (55:40:5 v/v %) and the elution was isocratic at a flow rate of 0.8 mL/min. Total analysis time was 12 min, and the detection of the tocols was performed with a fluorimetric detector where the excitation and emission wavelengths were set at 295 and 335 nm, respectively. Method validation was performed by means of intra-day (n = 5) and inter-day accuracy and precision (n = 8), sensitivity, and linearity. The linear regression coefficient (R2) was higher than 0.99. The recoveries of the tocols from barley samples with the proposed extraction method were in an acceptable level (74–91 %) where the relative standard deviation ranged from 4.2 to 15.0 %. Limits of detection (LODs) and limits of quantification (LOQs) varied from 0.03 to 0.11 mg kg−1 and 0.11 to 0.34 mg kg−1, respectively.

A survey on the occurrence of aflatoxin M1 in raw milk produced in Adana province of Turkey

During 2012, a total of 176 samples of raw milk obtained from dairy plants of Adana province of Turkey were analysed for the presence of aflatoxin M1 (AFM1). Aflatoxin M1 analysis was carried out by centrifugation, liquid–liquid extraction, immunoaffinity column clean-up and high performance liquid chromatography with fluorescence detection (HPLC-FD). The limits of detection (LOD) and quantification (LOQ) of the analytical method were 0.021 μg kg−1 and 0.025 μg kg−1. Accuracy of the method obtained from bias ranged from 2.94 to 8.70. Aflatoxin M1 was detected in 53 out of 176 samples analysed (30.1%). The ranges for positive samples were 0.042–0.552, 0.033–1.01, 0.047–0.150 and 0.025–0.102 μg kg−1 in autumn, winter, spring and summer seasons, respectively. Thirty samples of raw milk (17%) were above the legal limits of Turkey and EU regulations.

Characterization of newfound natural luminescent properties of melamine, and development and validation of a method of high performance liquid chromatography with fluorescence detection for its determination in kitchen plastic ware.

Experimental studies of the natural photoluminescence of melamine in aqueous solutions showed that its fluorescence intensity (at 250/365 nm) was appropriated for analytical purposes. The exploitation of such melamine property provided the basis of development of a new, simple, precise and accurate method based on high performance liquid chromatography with fluorescence detection (HPLC-Fluo) to determine melamine in kitchen plastic ware following aqueous extraction using a microwave oven. Optimization of analytical parameters such as solvent composition, pH and extraction conditions led to limits of detection and quantification of melamine of 0.0081 and 0.027 μg mL(-1), respectively, with a linear range up to 10 μg mL(-1). Sample extracts fortified with melamine at three concentration levels produced an average recovery of 98±6%, which was in agreement with the results achieved with a reference HPLC-UV method. Different samples of kitchen plastic ware analyzed by the developed and optimized method showed melamine concentrations in the aqueous extract up to 17 µg mL(-1), which corresponded to 86.0 mg kg(-1) in these utensils. The results obtained indicate that the use of kitchen plastic ware made of melamine can contaminate food with this compound after heating in a microwave oven.

Bioequivalence Study of Two 10 mg Montelukast Immediate-Release Tablets Formulations: A Randomized, Single-Dose, Open-Label, Two Periods, Crossover Study

tablets in twenty-four healthy volunteers. The Test product was Montelukast* manufactured by Tecnoquimicas S.A. (Jamundi – Colombia) and the Reference product was Singulair® (Montelukast) made by Merck Sharp & Dohme Ltd. (Northumberland - United Kingdom). A crossover design 2 x 2 of single-dose, with two treatments, two periods, two sequences and with a washout period of one week was used. Blood samples were collected from 0, 5 to 24 hours after dosing. The determination of Montelukast in plasma was performed using a previously validated bio-analytical method of high-performance liquid chromatography with fluorescence detector (HPLC-FLD). Through Montelukast concentration curves versus time measured in the volunteers’ plasma, the pharmacokinetic parameters and bioequivalence were determined for both products. The pharmacokinetic parameters determined in this study for both the reference and test products were Cmax 440.6 ± 227.4 ng/ml, 460.5 ± 170.9 ng/ml, AUC0→∞ 3196.5 ± 1546.8 ng/h.ml, 3284.9 ± 1270.0 ng/h.ml and AUC0→24 3162.5 ± 1537.6 ng/h.ml, 3251.6 ± 1221.8 ng/h.ml respectively. For Montelukast, with a confidence interval of 90%, the ratio of the logarithmic transformation test product / reference product for AUC0→∞ was from 94.5 to 110.9 and the ratio test product/reference product for Cmax was from 89.0 to 110.4. These intervals are within the established bioequivalence range and therefore determined that the test formulation is interchangeable or bioequivalent to the reference.

Presence of aflatoxins in cereals from Serbia

Aflatoxins (AFs), one of the most toxic and the strongest natural carcinogens can be found in a variety of food commodities, including cereals. For that purpose, the aim of this study was to investigate occurrence of AFs (AFB1, AFG1, AFB2 and AFG2) in 130 cereal samples. AFs content was determined by direct competitive Enzyme Linked Immunosorbent Assay (ELISA) method. Samples with AFs content higher than 1 μg/kg were analyzed again with confirmatory High Performance Liquid Chromatography with fluorescence detection (HPLC-FLD). Analyses showed that none of the analyzed wheat (30), barley (20), oats (20) and rye (20) samples was contaminated with AFs. On the other hand, among 40 analyzed maize samples 24 of them (60%) were contaminated in the following way: 6 (25%) samples had AFs concentration between 1 and 10 μg/kg, 14 (58%) samples between 10 and 50 μg/kg and 4 (17%) between 50 and 70.3 μg/kg. The most predominant aflatoxin was AFB1 which was detected in all contaminated maize samples. AFG1, AFB2 and AFG2 were found in 12, 5 and 1 sample, respectively. This study represents the first investigation of the occurrence of AFs in five different cereals from Serbia.

A comparative study of the analytical methods for the determination of polycyclic aromatic hydrocarbons in seafood by high‐performance liquid chromatography with fluorescence detection

SummaryThis study was performed to establish a rapid analytical method to determine the presence of fourteen polycyclic aromatic hydrocarbons (PAHs) in seafoods by high‐performance liquid chromatography with fluorescence detection (HPLC/FLD). The samples were prepared using two methods: the Quick, Easy, Cheap, Effective, Rugged, Safe (QuEChERS) method and the alkali digestion method. The QuEChERS method involved a convenient and effective solid–liquid extraction and a simple purification. The alkali digestion method was comprised of a liquid–liquid extraction after saponification with potassium hydroxide followed by purification. The limits of detection (LODs) and limits of quantification (LOQs) of the QuEChERS method ranged from 0.05 to 1.60 μg kg−1, and those of the alkali digestion method ranged from 0.28 to 5.18 μg kg−1. The repeatability for all target analytes was similar for the two methods, that is, 0.66–4.24% and 0.26–5.75% for the QuEChERS and alkali digestion methods, respectively. At analyte concentrations of 2.5–50 μg kg−1, the recovery of the QuEChERS method ranged from 86.87% to 115.67% and that of the alkali digestion method ranged from 69.22% to 100.21%.

Simultaneous determination of phenolic antioxidants in edible vegetable oils by HPLC–FLD assisted with second-order calibration based on ATLD algorithm

A novel strategy that combines the chemometrics method with high performance liquid chromatography with fluorescence detector (HPLC-FLD) was developed for the simultaneous determination of seven phenolic antioxidants in six kinds of oil samples. After a simple dilution step, oil samples can be directly injected into the detecting system and the data were measured in a short time with a chromatographic system operating in the gradient elution mode. Since the chromatographic and spectral peaks among interesting analytes and interferences were heavily overlapped, second-order calibration method based on alternating trilinear decomposition (ATLD) algorithm which fully exploiting the second-order advantage was adopted. Successful resolution was obtained in the presence of different matrix interferences in different oil samples, and the developed approach allows the quantification of the antioxidants at levels found in edible vegetable oils, without the necessity of applying either preconcentration or extraction steps, moreover, a column washing is also not required. Meanwhile, the effectiveness and reproducibility of the proposed method were also validated by some statistical parameters like root mean squared error of prediction (RMSEP), limits of detection (LOD) and relative standard deviation (RSD). Then the proposed method was compared with several commonly selected methods in sample preparation, elution time and LOD.

Enantioselective quantification of fluoxetine and norfluoxetine by HPLC in wastewater effluents

Microbial degradation is the most important process to remove organic pollutants in Waste Water Treatment Plants. Regarding chiral compounds this process is normally enantioselective and needs the suitable analytical methodology to follow the removal of both enantiomers in an accurate way. Thus, this paper describes the development and validation of an enantioselective High Performance Liquid Chromatography with Fluorescence Detection (HPLC-FD) method for simultaneous analysis of fluoxetine (FLX) and norfluoxetine (NFLX) in wastewater effluents. Briefly, this method preconcentrated a small volume of wastewater samples (50mL) on 500mg Oasis MCX cartridges and used HPLC-FD with a vancomycin-based chiral stationary phase under reversed mode for analyses. The optimized mobile phase was EtOH/aqueous ammonium acetate buffer (92.5/7.5, v/v) at pH 6.8. The effect of EtOH percentage, buffer concentration, pH, column oven temperature and flow rate on chromatographic parameters was systematically investigated. The developed method was validated within the wastewater effluent used in microcosms laboratory assays. Linearity (R2>0.99), selectivity and sensitivity were achieved in the range of 4.0–60ngmL−1 for enantiomers of FLX and 2.0–30ngmL−1 for enantiomers of NFLX. The limits of detection were between 0.8 and 2.0ngmL−1 and the limits of quantification were between 2.0 and 4.0ngmL−1 for both enantiomers of FLX and the enantiomers of its demethylated metabolite NFLX. The validated method was successfully applied and proved to be robust to follow the degradation of both enantiomers of FLX in wastewater samples, during 46days.

Multi-mycotoxin determination in baby foods andin vitro combined cytotoxic effects of aflatoxin M1 and ochratoxin A

The co-occurrence of mycotoxins in baby foods, including aflatoxin M1(AFM1) and ochratoxin A (OTA), has been reported in previous studies, but data on their potential combined toxic effects are still missing. The present work aimed at (1) validating an in-house multi-mycotoxin high performance liquid chromatography with fluorescence detection (HPLC-FLD) method for AFM1, total aflatoxins (aflatoxin B1(AFB1), aflatoxin B2(AFB2), aflatoxin G1(AFG1), aflatoxin G2(AFG2)) and OTA in infant formulae (milk powders) and cereal baby foods (flours), and (2) assessing the combined cytotoxic effects of AFM1and OTA in an intestine-derived cell line. The HPLC-FLD method, which included a chloroform extraction, liquid-liquid extraction, immunoaffinity column clean-up and fluorescence detection after post-column derivatisation with electrochemically generated bromine, was adequate for the analysis of baby foods and met the requirements of validation and quality control for the studied working ranges. The limits of quantification for AFM1, AFB1, AFB2, AFG1, AFG2and OTA were 0.069, 0.032, 0.020, 0.047, 0.020 and 0.244 μg/kg, respectively. The mean recovery values were 96, 114, 112, 107, 101 and 87%, respectively. A dose-dependent cytotoxicity was observed for individual and combined AFM1and OTA using the Caco-2 cell line, which represents a site of contact of both mycotoxins in the body, after oral exposure. Interactions between both mycotoxins were disclosed by application of the concentration addition (CA) and independent action (IA) models, revealing the predominance of an antagonistic pattern. In conclusion, this study proposes a HPLC-FLD method for multi-mycotoxin monitoring in baby foods and provides evidence for the interaction between AFM1and OTA, and for the applicability of CA/IA models to predict the effect of mycotoxins mixtures, further contributing to the prevention of mycotoxins-associated adverse health effects.

Tracing the origin of paralytic shellfish toxins in scallop Patinopecten yessoensis in the northern Yellow Sea

Some dinoflagellate species within the genera Alexandrium, Gymnodinium and Pyrodinium are well-known producers of paralytic shellfish toxins (PST), which led to many poisoning incidents around the world. In the northern Yellow Sea, an important mariculture zone for scallop Patinopecten yessoensis, PST have been frequently detected from scallops. However, there is little knowledge concerning PST-producing microalgae in this region so far. In cruises carried out in 2011 and 2012, scallop and phytoplankton samples were collected from the northern Yellow Sea. PST were detected from scallops by high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Toxin content and profile were remarkably different among the four tissues, i.e. viscera, adductor muscle, mantle and gonad, suggesting apparent toxin transfer and transformation in scallops. Viscera always had the highest content of PST dominated by low-potency N-sulfocarbamoyl toxins C1 and C2, which closely resembled the toxin profiles of net-concentrated phytoplankton samples in spring. Based on the morphological features, cells of Alexandrium spp. in net-concentrated phytoplankton samples were picked out and a partial sequence of the large subunit ribosomal RNA gene (LSU rDNA) was amplified using a single-cell polymerase chain reaction (PCR) method. Cells of both toxic A. tamarense species complex and non-toxic A. affine were identified from the phytoplankton samples based on the partial LSU rDNA sequence information. According to these findings, it is implied that A. tamarense species complex is the major toxic species related to PST contamination in scallops of the northern Yellow Sea. The presence of both toxic and non-toxic Alexandrium spp. in this region requires for a species-specific method to monitor the distribution and dynamics of A. tamarense species complex.

Development of a Precolumn Derivatization Procedure Prior to Ultrasound-Assisted Cloud Point Extraction for Sensitive Determination of Fluoroquinolones in Eggs by High-Performance Liquid Chromatography with Fluorescence Detection

In this work, a 4-chloro-7-nitrobenzodioxazole (NBD-Cl) derivatization coupled with the ultrasound-assisted cloud point extraction (UACPE) method prior to high-performance liquid chromatography with fluorescence detection (HPLC-FLD) analysis was developed for the determination of four widely used fluoroquinolones (FQs) including norfloxacin (NOR), ciprofloxacin (CIP), sarafloxacin (SAR), and gatifloxacin (GAT) in eggs. The derivatives of FQs with NBD-Cl were extracted into the Triton X-114 surfactant-rich phase, which was analyzed by reversed-phase HPLC-FLD. Ultrasound was applied to accelerate the phase separation in extraction and enhance the extraction efficiency of target analytes. Variable parameters affecting the derivatization and UACPE procedure were systematically evaluated and optimized. Under the optimum conditions, four FQs were successfully separated within 30 min through an Agilent TC-C18 column. Good recoveries of 86.2–103.5 %, which were calculated using a range of spiked samples at three concentrations, were obtained by UACPE. The calibration graphs were linear over the range of 1.2–73.0 μg kg−1 for four FQs with correlation coefficients (R) no less than 0.9957. The limits of detection were 0.2, 0.5, 0.3, and 0.4 μg kg−1 for NOR, CIP, SAR, and GAT, respectively. The precisions indicated by relative standard deviations ranged from 0.6 to 4.3 % for both intraday and interday analysis. The proposed method proved to be a selective, sensitive, and eco-friendly approach which was successfully applied to analyze FQs in eggs at the local farmers market, and none of the target analytes were detected in these samples.

Biogenic amines in commercially produced Yulu, a Chinese fermented fish sauce

Seven biogenic amines were determined in 35 commercially produced Yulu samples from three provinces of China by pre-column derivatisation with dansyl chloride (Dns-Cl) and high-performance liquid chromatography with fluorescence detection (HPLC-FLD). Putrescine, cadaverine, histamine and tyramine were the major biogenic amines (more than 100 mg kg−1), while tryptamine, spermidine and spermine were regarded as minor biogenic amines (less than 25 mg kg−1). Twenty samples contained more than 50 mg kg−1 histamine (the limit for histamine in seafood products as suggested by the Food and Drug Administration). Twenty-one samples contained more than 100 mg kg−1 tyramine and 10 contained more than 1000 mg kg−1 total biogenic amines. This study provided data on biogenic amine levels in Chinese fermented fish sauce. The results suggested that biogenic amine content should be monitored in commercially produced Yulu.