Validation of a multi-residue enzyme-linked immunosorbent assay for qualitative screening of corticosteroids in liver,urine and milk

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was applied for the qualitative screening analysis of dexamethasone, betamethasone, flumethasone, and prednisolone in milk and urine, and dexamethasone, flumethasone and prednisolone in liver samples at levels corresponding to the European Union maximum residue limit (MRL), or at required performance levels (RPLs) for substances for which there is no established MRL. Method validation was performed according to Commission Decision 2002/657/EC criteria established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, limit of detection (LOD), limit of quantitation (LOQ), recovery, within-laboratory reproducibility, linearity and ruggedness. LODs were 0.2, 1.2 and 0.6?µg?kg^?1 in milk, urine and liver samples, and LOQ values were 0.3, 1.2 and 1.4?µg?kg^?1 in milk, urine and liver, respectively. Recoveries from spiked samples ranged from 68% to 131% for dexamethasone, from 57% to 120% for flumethasone, from 60% to 155% for betamethasone, and from 23% to 32% for prednisolone, with a coefficient of variation (CV) between 1.6% and 21.2%. The CCβ value was below the MRL/RPL for all examined matrices. Moderate variations of some critical factors in the sample pre-treatment for liver and milk samples were deliberately introduced for ruggedness evaluation and did not result in any negative effects on corticosteroid detection. The proposed method is suitable for qualitative screening analysis of corticosteroids in the above-mentioned food in conformity with the current European Union performance requirements.     

Screening and quantification of 18 glucocorticoid adulterants from herbal pharmaceuticals and health foods by HPLC and confirmed by LC-Q-TOF-MS/MS

A procedure for the screening and quantification of 18 glucocorticoids, i.e. hydrocortisone sodium succinate (HSS), prednisolone (PDL), prednisone (PDS), hydrocortisone (HCS), methylprednisolone (MPS), betamethasone (BTM), dexamethasone (DXM), triamcinolone acetonide (TA), prednisolone acetate (PLA), hydrocortisone acetate (HA), fludrocortisone acetate (FA), prednisone acetate (PA), cortisone acetate (CA), dexamethasone acetate (DA), hydrocortisone butyrate (HB), triamcinolone acetonide acetate (TAA), fluocinonide (FN) and halcinonide (HC) from herbal pharmaceuticals and health foods was established and fully validated. The samples were extracted by methanol and separated by HPLC. The retention times and ultraviolet spectra were used for the preliminary screening, and the suspected adulterants were then confirmed by liquid chromatography-quadrupole-time of flight mass spectrometry (LC-Q-TOF-MS/MS) and quantified by HPLC. The developed procedure was successfully applied to 14 herbal samples, and 316.3 µg g^–1 of DA and 13.6 µg mL^–1 of BTM were found in a tablet sample and a spray sample, respectively. To our best knowledge, this is the first report of the simultaneous screening and quantification of these 18 glucocorticoids from any matrix.     

Development of a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of glucocorticoid residues in edible tissues of swine,cattle, sheep,and chicken

A confirmatory and quantitative method using liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine the presence of eight glucocorticoids (prednisone, prednisolone, hydrocortisone, methylprednisolone, dexamethasone, betamethasone, beclomethasone, and fludrocortisone) in the muscles and livers of swine, cattle, and sheep and the muscle of chicken is described. After deconjugation in alkali media, samples were extracted with ethyl acetate for glucocorticoids followed by solid-phase extraction clean-up and reconstitution in the LC mobile phase. The hydrolysis procedure with sodium hydroxide was used to reduce handling time. A single-step solid-phase extraction method was optimized which is suitable for the clean-up of the compounds of interest in many diverse tissue matrices. LC separations were performed on a C₁₈ column with gradient elution using acetonitrile and water (containing 0.2% formic acid) and the two epimers betamethasone and dexamethasone were successfully separated. LC-electrospray ionization (ESI)-MS/MS in negative mode with selected reaction monitoring (SRM) mode was performed to improve method sensitivity and reduce matrix interference. Two SRM transitions were used for each compound. The recovery of glucocorticoids spiked at levels of 0.5–16 µg kg^?1 ranged from 55% to 107%; the between-day relative standard deviations were no more than 15%. The limits of quantification were 0.5–2.0 µg kg^?1 in muscle and 1–4 µg kg^?1 in liver. The optimized procedure was successfully applied to monitor the food at the 2008 Summer Olympics Games in Beijing, China, demonstrating the method to be simple, fast, robust, and suitable for identification and quantification of glucocorticoids residues in foods of animal origin.     

Validation of a multi-residue enzyme-linked immunosorbent assay for qualitative screening of corticosteroids in liver, urine and milk

A rapid and sensitive enzyme-linked immunosorbent assay (ELISA) was applied for the qualitative screening analysis of dexamethasone, betamethasone, flumethasone, and prednisolone in milk and urine, and dexamethasone, flumethasone and prednisolone in liver samples at levels corresponding to the European Union maximum residue limit (MRL), or at required performance levels (RPLs) for substances for which there is no established MRL. Method validation was performed according to Commission Decision 2002/657/EC criteria established for qualitative screening methods. In this regard, the following parameters were determined: detection capability (CCβ), specificity, limit of detection (LOD), limit of quantitation (LOQ), recovery, within-laboratory reproducibility, linearity and ruggedness. LODs were 0.2, 1.2 and 0.6 μg kg(-1) in milk, urine and liver samples, and LOQ values were 0.3, 1.2 and 1.4 μg kg(-1) in milk, urine and liver, respectively. Recoveries from spiked samples ranged from 68% to 131% for dexamethasone, from 57% to 120% for flumethasone, from 60% to 155% for betamethasone, and from 23% to 32% for prednisolone, with a coefficient of variation (CV) between 1.6% and 21.2%. The CCβ value was below the MRL/RPL for all examined matrices. Moderate variations of some critical factors in the sample pre-treatment for liver and milk samples were deliberately introduced for ruggedness evaluation and did not result in any negative effects on corticosteroid detection. The proposed method is suitable for qualitative screening analysis of corticosteroids in the above-mentioned food in conformity with the current European Union performance requirements.     

Development of an immunochromatographic assay for the β-adrenergic agonist feed additive zilpaterol

Zilpaterol is a β-adrenergic agonist feed additive approved in the United States to increase weight gain and improve feed efficiency of cattle. A zilpaterol immunochromatographic assay was developed as an economical and user-friendly rapid detection method for zilpaterol and validated using urine and tissue samples derived from animal studies. The assay sensitivity was 1.7–23.2 ng g^?1 or mL^?1 across a variety of feed and animal matrices and did not cross-react with clenbuterol or ractopamine. No sample pre-treatment of cattle and sheep urine was needed, but horse urine and feed required dilution; skeletal muscle required solvent extraction prior to testing. Of 32 incurred sheep urine samples tested, zilpaterol content was correctly identified in all but 2 samples. Horse urine containing >10 ng mL^?1 of incurred zilpaterol residue (n = 48) was correctly identified as zilpaterol positive. The assay correctly identified 0-day withdrawal sheep muscle samples as zilpaterol positive and the control and longer withdrawal day sheep muscle samples as negative. Zilpaterol was demonstrated to be stable in horse urine when stored at ?20°C for 7 years.     

Aflatoxins in sunflower seeds and unrefined sunflower oils from Singida,Tanzania

A total of 61 samples comprising sunflower seeds (40) and unrefined sunflower oils (21) samples collected randomly from Singida, Tanzania were analysed by Reverse Phase-high performance liquid chromatography (RP-HPLC). 15% (6/40) of the seed samples were contaminated with aflatoxin B₁ ranging from limit of detection (LOD) to 218 ng g^?1 with three of them exceeding the European Commission/European Union (EC/EU) and Tanzania Bureau of Standards (TBS)/Tanzania Food and Drug Authority (TFDA) maximum limits of 2 ng g^?1 for AFB₁ in oilseeds. The levels of total aflatoxins (AFT) in seeds ranged from LOD to 243 ng g^?1. Other aflatoxins, except AFG₂, were also detected. For the unrefined sunflower oils, the levels of AFB₁ ranged from LOD to 2.56 ng mL^?1. About 80.9% (17/21) of the analysed oil samples contained AFB₁ of which 17.65% (3/17) exceeded the EC/EU and TBS/TFDA maximum limits of 2 ng mL^?1. Other aflatoxins were also detected in the oils. The measured levels indicate there is a need for food quality education among food processors.     

Aflatoxin M1 in raw milk and aflatoxin B1 in feed from household cows in Singida,Tanzania

Aflatoxin M₁ (AFM₁) contamination in raw milk from household cows fed with sunflower seedcakes or sunflower-based seedcake feeds was determined in 37 milk samples collected randomly from different locations in Singida region, Tanzania. Aflatoxin B₁ (AFB₁) contamination in sunflower-based seedcake feed was determined in 20 feed samples collected from the same household dairy farmers. The samples were analysed by RP-HPLC using fluorescent detection after immunoaffinity column clean-up. Recoveries were 88.0% and 94.5%, while the limits of detection (LOD) were 0.026 ng mL^?1 and 0.364 ng g^?1 for AFM₁ and AFB₁, respectively. Of the analysed cow’s milk samples, 83.8% (31/37) contained AFM₁, with levels ranging from LOD to 2.007 ng mL^?1, exceeding both the European Commission (EC) and Tanzania Food and Drug Authority (TFDA) limit of 0.05 ng mL^?1. Of the contaminated samples, 16.1% exceeded the Codex Alimentarius limit of 0.5 ng mL^?1. AFB₁ was present in 65% (13/20) of the feed samples with levels ranging from LOD to 20.47 ng g^?1, 61.53% exceeding the TFDA and EC maximum limits of 5 ng g^?1 for complete dairy animal feed. The observed AFM₁ and AFB₁ contamination necessitates the need to raise awareness to dairy farmers in Tanzania to safeguard the health of the end-users.     

Corticosteroids in liver and urine in Sicilian cattle by a LC-MS/MS method

The presence of corticosteroid residues was assessed in urine and liver samples from livestock of Sicily. A total of 630 bovine samples were collected from farms and slaughterhouses. The samples were analysed using solid-phase extraction (SPE) coupled with ultra-performance liquid chromatography tandem mass spectrometry (UHPLC–MS/MS). All the corticosteroids found were under the maximum residue limit imposed by Commission Regulation (EC) 37/2010. About 4% of liver samples showed dexamethasone levels above the limit of detection (LOD), with a mean of 1.5 ± 0.2 µg kg^?1. Betamethasone was found only in seven liver samples, with a mean of 1.6 ± 0.1 µg kg^?1. Furthermore, prednisolone and prednisone were found only in urine and liver samples from slaughterhouse, probably related to the high rate of stress for bovines. These results suggest good control practices adopted by Sicilian farms, able to ensure the quality of food products.     

A Preconcentration Procedure for Determination of Ultra-Trace Mercury (II) in Environmental Samples Employing Continuous-Flow Cold Vapor Atomic Absorption Spectrometry

New functionalized magnetic nanoparticles as solid-phase sorbent were prepared and investigated for extraction of ultra-trace amounts of mercury from environmental samples. The Fe₃O₄ magnetic nanoparticles functionalized with dithizone were characterized by Fourier transform infrared spectrometer. X-ray diffraction and scanning electron microscopy confirmed the size of nanoparticles. Effects of several factors on the extraction procedure were investigated. The optimized conditions were established to be 80 mg of polymer, 8.5 for solution pH, 5 min for adsorption time, 5 min for desorption time, 2 mL for HCl (0.1 mol L^?1)/ thiourea 0.05 % as the eluent, 500 mL for breakthrough volume, and without addition of salt. Under the optimal conditions, the limit of detection, maximum capacity, and preconcentration factor were 0.05 ng mL^?1, 0.557 mmol g^?1, and 250, respectively. Limit of quantification was in the range of 0.2–2 ng mL^?1 for various matrices. Accuracy and precision of the method were about ±2.0 and below 11.1 %, respectively. Finally, the present method has been successfully applied to mercury determination in table salt, green tea, vegetables, toothpaste, and water samples. The mercury content found in the real samples was from 0.6 to 15.74 ng mL^?1 without addition of mercury.     

Urinary aflatoxin exposure monitoring in rural and semi-urban populations in Ogun state,Nigeria

Aflatoxins are a major class of fungal toxins that have food safety importance due to their economic and health impacts. This pilot aflatoxin exposure biomonitoring study on 84 individuals was conducted in a rural (Ilumafon) and a semi-urban community (Ilishan Remo) of Ogun state, Nigeria, to compare aflatoxin exposures among the two population cohorts. First morning urine samples were obtained from the participants, and the urinary aflatoxin M₁ (AFM₁) levels were measured by a quantitative Helica Biosystems Inc. ELISA kit assay. About 99% (83 out of 84) of the urine samples had detectable AFM₁ levels in the range of 0.06 to 0.51 ng mL^?1 (median: 0.27 ng mL^?1). The mean urinary AFM₁ levels were significantly (p = 0.001) higher in the semi-urban population (0.31 ± 0.09 ng mL^?1) compared to the rural population (0.24 ± 0.07 ng mL^?1). There were, however, no significant differences in mean urinary AFM₁ levels of males and females, and among children, adolescents and adults. This study indicates high aflatoxin exposure to the extent of public health concerns in the studied populations. Thus, more efforts are required for aflatoxin exposure monitoring and control in high-risk regions.     

A Novel Method for Bisphenol A Analysis in Dairy Products Using Graphene as an Adsorbent for Solid Phase Extraction Followed by Ion Chromatography

A novel and reliable ion chromatography (IC) method using graphene (G) as a solid phase extraction (SPE) adsorbent for the rapid analysis of bisphenol A (BPA) in dairy products was developed. The performances of graphene (G) and commercial C18 for BPA extraction from dairy samples were evaluated; results showed that G had higher adsorption efficiency. IC coupled with an electrochemical detector (ED) is eco-friendly, labor and time saving compared to liquid chromatography mass spectrometry (LC-MS) and gas chromatography mass spectrometry (GC-MS). The effects of the experimental parameters of the IC-ED system were assessed, and the parameters were optimized to provide maximum sensitivity. The linear range is 5–20,000 ng?mL^?1 with an R value of 0.999. The limit of the detection is 0.8 ng?mL^?1 for a 25-μL injection loop. The mean relative recoveries ranged between 83.3 % and 104.6 %, the corresponding inter-day precision was below 5.3 % for 20, 200, 2,000, and 15,000 ng?mL^?1. This method was successfully employed to analyze BPA in dairy samples.     

A field survey on the presence of prednisolone and prednisone in urine samples from untreated cows

Prednisolone is a synthetic glucocorticoid widely employed in bovine clinical practice that may also be used illegally as a growth promoter. Recent in vitro and in vivo studies lend support to the hypothesis that prednisolone could be synthesised from cortisol in untreated cattle subjected to stressful events. To verify such a hypothesis, a field survey was conducted on urine samples collected from 131 guaranteed untreated cows and analysed for the presence of prednisolone and prednisone – in some instances also for cortisol and cortisone – with a validated LC/MS-MS method. None of the examined samples exhibited either prednisolone levels higher than the CCα limit (around 0.70?µg l^?1) or prednisone, being therefore officially compliant for both analytes. Trace amounts of prednisolone, approximately estimated in the range 0.1–0.3?µg l^?1 were found in only seven samples from cows also showing urinary cortisol and cortisone levels higher than those detected in negative specimens, as the result of a probable stress condition.     

Studies on the Aggregation-Induced Synchronous Emission of 1,8-Naphthalimide Derivative to Casein and Its Analytic Application

A novel water-soluble 1,8-naphthalimide probe 1, bearing two acetic carboxylic groups, exhibited high selectivity and sensitivity for recognition of casein with the aggregation-induced synchronous emission, based on which, a new casein assay method was developed. This method exhibited a good linear range from 0.1 to 20.0 μg?mL^?1 and 0.1 to 16.0 μg?mL^?1, with the correlation coefficient of 0.9975 and 0.9982. The detection limits were estimated to be 4.5 and 6.7 ng?mL^?1. The proposed method was applied to the determination of casein in milk powder samples and the results were in good agreement with the result of Buiret method.     

Effect-based detection of synthetic glucocorticoids in bovine urine

Challenges to testing for the illicit use of anabolic substances in meat-producing animals stem from the production of new synthetic compounds and the administration of low-dose cocktails to circumvent detection by the surveillance schemes of European Union member states. This work evaluated for the first time GR-CALUX, a highly sensitive reporter gene assay, as a screening tool for the detection of synthetic glucocorticoids in bovine urine. In order to verify the effect of natural corticosteroids on the method, the bioassay was tested first using blank urine samples collected at the farm and the slaughterhouse. Next, the dose–response curves were measured for the most commonly used synthetic glucocorticoids. The bioassay’s ability to detect them in spiked and incurred samples of bovine urine was then evaluated. Finally, its performance was compared against a commercially available ELISA kit ordinarily used in screening activities. GR-CALUX performance did not appear to be influenced by physiological levels of endogenous corticosteroids in the farm samples, whereas an increase in these hormones might invalidate the analysis in samples obtained at the slaughterhouse. Using pure compounds, GR-CALUX showed a high sensitivity toward the synthetic glucocorticosteroids tested in order of relative potencies: flumethasone ? dexamethasone > betamethasone > methylprednisolone > prednisolone. As expected, the bioassay failed to detect the prohormone prednisone. The results obtained from analysis of the spiked and incurred specimens reproduced those of the blank samples and the pure compounds. GR-CALUX is a promising screening tool for the detection of illicit treatments in meat-producing bovines. Its ability to detect the most commonly used synthetic glucocorticoids was comparable with the ELISA test. Importantly, it appeared to be less susceptible to matrix effects than ELISA.     

3-Monochloro-1,2-propandiol (3-MCPD) in soy sauce from the Bulgarian market

The 3-monochloro-1,2-propandiol (3-MCPD) levels in soy sauces which contained hydrolysed vegetable protein were evaluated for the Bulgarian market. For analysis of 3-MCPD, a gas chromatography–mass spectrometry (GC-MS) method was applied with a linear range of 0.03–2.00 μg mL^?1 and a limit of detection (LOD) of 2.3 μg kg^?1 and a limit of quantification (LOQ) of 3.4 μg kg^?1. At these levels, the standard deviation was 5.1%, with recoveries between 81% and 102%. The method was applied to the analysis of 21 samples of soy sauce from the Bulgarian market. Results ranged from 3.7 to 185.6 μg kg^?1. Soy sauces produced from hydrolysed soy protein contained higher levels of 3-MCPD than naturally fermented sauces. In 38.4% of samples of Bulgarian origin, the 3-MCPD content was above the EU limit of 20 μg kg^?1. In all analysed samples, 33.3% had a 3-MCPD content above the EU limit.     

Investigation of the presence of prednisolone in bovine urine

Over the past two years low levels of prednisolone have been reported in bovine urine by a number of laboratories in European Union member states. Concentrations vary, but are reported to be below approximately 3 µg l^–1. Forty per cent of bovine urine samples from the Dutch national control plan had concentrations of prednisolone between 0.11 and 2.04 µg l^–1. In this study the mechanism of formation of prednisolone was investigated. In vitro conversion of cortisol by bacteria from faeces and soil, bovine liver enzymes and stability at elevated temperatures were studied. In vitro bovine liver S9 incubation experiments showed a significant 20% decrease of cortisol within 6 h, and formation of prednisolone was observed from 0.2 g l^–1 at t = 0 to 0.5 g l^–1 at t = 6. Under the influence of faeces, the stability of cortisol in urine is reduced and cortisol breaks down within 50 h. Prednisolone is formed up to 4 µg l^–1 at 70°C after 15 h. However, this decreases again to zero after 50 h. With soil bacteria, a slower decrease of cortisol was observed, but slightly higher overall formation of prednisolone, up to 7 µg l^–1 at 20°C. As opposed to incurred urine, in fortified urine incubated with faeces or soil bacteria no prednisolone was detected. This difference may be explained by the presence of natural corticosteroids in the incurred sample. With UPLC-QToF-MS experiments, in urine and water samples incubated with faeces, metabolites known from the literature could be (tentatively) identified as 20β-hydroxy-prednisolone, cortisol-21-sulfate, oxydianiline, tetrahydrocortisone-3-glucuronide and cortexolone, but for all compounds except 20β-hydroxy-prednisolone no standards were available for confirmation. Based on the results of this study and literature data, for regulatory purposes a threshold of 5 µg l^–1 for prednisolone in bovine urine is proposed. Findings of prednisolone in concentrations up to 5 µg l^–1 in bovine urine can, most likely, originate from other sources than illegal treatment with growth promoters.     

Simultaneous Determination of Cyclamate,Acesulfame, and Aspartame in Beverages by Titania-Based RP-HPLC

This paper aims at establishing a rapid method for simultaneous separation and determination of the sweeteners including cyclamate, acesulfame, and aspartame in beverages by titania-based reversed-phase high-performance liquid chromatography. The chromatographic conditions were as follows: a titania Sachtopore-RP C18 column (250?×?4.6 mm; 5 μm) as a separation column, a mixture of water and methanol at a volume ratio of 95:5 containing 1.0 % phosphate acid used as mobile phase, flow rate of 1.0 mL min^?1, and the detection wavelength was 205 nm. The linear ranges of cyclamate and aspartame were 0.02–8.0 mg mL^?1 with limits of detection (LODs) of 16.35 and 19.56 μg mL^?1, respectively, and their limits of quantitation (LOQs) were 55.50 and 68.50 μg mL^?1, respectively. The recoveries of cyclamate were between 93.52 and 103.54 %. The recoveries of aspartame were between 93.31 and 102.63 %. The linear range of acesulfame was 0.125–50 μg mL^?1 with LOD of 0.08 μg mL^?1 and LOQ of 0.25 μg mL^?1. The recoveries of acesulfame were between 94.34 and 103.21 %. Relative standard deviation (n?=?8) for all determinations was less than 0.72 %.     

Immunobiosensor determination of β-agonists in urine using integrated immunofiltration clean-up

A fast immunobiosensor assay for screening of β‐agonists in urine, using integrated immunofiltration clean‐up with 30 kDa cutoff centrifugal filter, was developed. The same antibody aliquot was used for clean‐up and biosensor analysis, and no organic solvent was needed. Matrix interference from urine was efficiently reduced. Two antibodies with different cross‐reactivities were compared. Although both antibodies were able to detect clenbuterol (CBL) below 1 ng mL^?1, a polyclonal antibody directed against salbutamol was chosen because of its ability to recover several β‐agonists. The decision limit (CCα) of the final assay was 0.29 ng mL^?1, and an α‐error of 1% gave a β‐error of <1% for twenty reference bovine samples spiked with 1 ng mL^?1 CBL.     

Quantitative Determination and Confirmation of Five Synthetic Glucocorticoid Residues in Milk Powder by Gel Permeation Chromatography–Liquid Chromatography–Tandem Mass Spectrometry

A new method for multi-residue determination of five synthetic glucocorticoids (betamethasone, dexamethasone, prednisone, prednisolone, and hydrocortisone) in milk powder is developed. The glucocorticoids in the samples were extracted with tert-butyl methyl ether under ultrasonication incubation, and then cleaned up through gel permeation chromatography. After C18 LC gradient elution separation using acetonitrile with 0.1% formic acid as a mobile phase, the eluents were determined by liquid chromatography–tandem mass spectrometry with multiple reaction monitoring and positive ionization modes. The effective separation for the five glucocorticoids was achieved. The limit of quantification of the method for testing five glucocorticoids in milk powder was in the range from 0.2 to 0.5 μg kg⁻¹, which was lower than the maximum residue limits established by European Union for glucocorticoids in foods. Experiments on spiked samples of milk powder showed that the mean recoveries at addition level of 1.0, 3.0, and 5.0 μg kg⁻¹ were in the range of 71.2% and 103%, and the relative standard deviation ranged from 4.7% to 16.8%. The calibration curves for these drugs between 1.0 and 100 μg l⁻¹ showed good linearity, with correlation coefficient (r) more than 0.999. The real sample test showed that this method is sensitive and accurate. It can be used for qualitative and quantitative determination of studied glucocorticoid residues in milk powder samples.     

Development of a sensitive polyclonal antibody-based competitive indirect ELISA for determination of citrinin in grain-based foods

ABSTRACT

A sensitive competitive indirect enzyme-linked immunosorbent assay (ciELISA) was developed for the detection and quantification of citrinin (CIT) in grain-based food samples. The limit of quantification (IC₂₀) of the established method was 0.10 ± 0.02 ng mL^?1, with the limit of detection (IC₁₀) being 0.04 ± 0.007 ng mL^?1 in wheat and corn flour matrices with a coefficient of variation (CV) less than 20%. The assay was very specific to CIT and showed no cross-reactivity with other mycotoxins (OTA, T-2 toxin, HT-2 toxin, DON, patulin and zearalenone). In spiked wheat and corn flours, the recoveries ranged from 86.6% to 115.6% with CVs of less than 20%. The effectiveness of this method was verified by participating in a proficiency test (PT) from the Food Analysis Performance Assessment Scheme (FAPAS) 17181 corn flour. A successful z-score (?0.6) for this PT sample showed that the present method is comparable to the instrumental methods used by other laboratories in the PT testing scheme. A small survey of grain-based foods was conducted using this method and CIT was detected in 43% of the samples up to a concentration of 17.7 ng g^?1. This method is suitable for sensitive and rapid quantitation of citrinin in wheat and corn matrices.