Screening on the occurrence of ochratoxin A in green coffee beans of different origins and types

Since to our knowledge no data are available in the literature regarding the influence of green coffee type and origin on ochratoxin A (OTA) content, determinations were carried out in order to assess the level of OTA contamination in green coffee samples of different provenience. A total of 162 samples of green coffee beans from various countries (84 from Africa, 60 from America, and 18 from Asia) were analyzed for OTA. Both the amount and the variability of OTA levels were tested as a function of green coffee provenience. The results showed that 106 of the overall samples were positive for OTA, with concentration ranging from 0 to 48 microg/kg (ppb). In particular, it was possible to verify that African samples were more contaminated with respect to samples of other origin in terms of frequency and level of OTA; the highest concentrations observed were 18 and 48 microg/kg in two samples from The Congo.     

Analysis of ochratoxin a in coffee by solid-phase cleanup and narrow-bore liquid chromatography-fluorescence detector-mass spectrometry

A method for the analysis of ochratoxin A (OTA) in green and roasted coffee has been developed. OTA was extracted from coffee with 1% NaHCO(3), and the extract was filtered and purified by solid-phase cleanup using a polymeric column that exhibits reversed-phase and anion-exchange functionalities. OTA was analyzed on a narrow-bore reversed-phase C(18) HPLC column with acetonitrile/water (0.1% formic acid) (40:60) as mobile phase and quantified with a fluorescence detector. The presence of OTA in coffee was confirmed by single-quadruple mass spectrometry using an electrospray ionization source. The method has been validated, obtaining a recovery of 82.5% and a detection limit of 0.1 ng/g. It has been applied to 20 coffee samples from various countries and different manufacturers with no detection of OTA.     

Development of a solid-phase cleanup and portable rapid flow-through enzyme immunoassay for the detection of ochratoxin a in roasted coffee

A membrane-based flow-through enzyme immunoassay (patent application pending) for the detection of ochratoxin A (OA) in roasted coffee was developed. First, an extraction and solid-phase cleanup method was developed. A high partition coefficient for OA in the mobile phase was achieved by using methanol/5% aqueous NaHCO(3) as the sample extraction and cleanup solvent. The solid-phase (aminopropyl) cleanup was developed to chromatographically elute OA but retain cross-reacting compounds. Without using aminopropyl cleanup, cross-reacting compounds resulted in 100% false positives for both flow-through enzyme immunoassay and HPLC methods. However, after cleanup with aminopropyl, no false positives were observed. The flow-through results were visually evaluated. The sensitivity achieved for the flow-through was 4 microg kg(-1) in spiked roasted coffee. The assay was used to screen roasted coffee samples. Results were confirmed with HPLC with a detection limit of 1 microg kg(-1).     

Occurrence of ochratoxin A in cocoa products and chocolate

In this work, the occurrence of ochratoxin A (OTA) in 170 samples of cocoa products of different geographical origins was studied. An immunoaffinity column with HPLC separation was developed to quantify low levels of OTA in cocoa bean, cocoa cake, cocoa mass, cocoa nib, cocoa powder, cocoa shell, cocoa butter, chocolate, and chocolate cream with >80% recoveries. The method was validated by performing replicate analyses of uncontaminated cocoa material spiked at three different levels of OTA (1, 2, and 5 microg/kg). The data obtained were related on the acceptable safe daily exposure for OTA. The highest levels of OTA were detected in roasted cocoa shell and cocoa cake (0.1-23.1 microg/kg) and only at minor levels in the other cocoa products. Twenty-six cocoa and chocolate samples were free from detectable OTA (<0.10 microg/kg). In roasted cocoa powder 38.7% of the samples analyzed contained OTA at levels ranging from 0.1 to 2 microg/kg, and 54.8% was contaminated at >2 microg/kg (and 12 samples at >3 microg/kg). Ochratoxin A was detected in cocoa bean at levels from 0.1 to 3.5 microg/kg, the mean concentration being 0.45 microg/kg; only one sample exceeded 2 microg/kg (4.7%). In contrast, 51.2% of cocoa cake samples contained OTA at levels > or =2 microg/kg, among which 16 exceeded 5 microg/kg (range of 5-9 microg/kg). These results indicate that roasted cocoa powder is not a major source of OTA in the diet.     

Evaluation of methods used to determine ochratoxin A in coffee beans

A comparative study was conducted to evaluate four previously reported methods that proved to have a recovery greater than 80% for the determination of different levels of ochratoxin A (OTA) in green and roasted coffee beans and to select an accurate, sensitive, and less-expensive technique between the existing methods. The results indicated that the Association of Official Analytical Chemists (AOAC) official method for the extraction of OTA in green coffee and determination by high-performance liquid chromatography (HPLC) is recommended as an efficient method for the routine analyses of OTA in green and ground roasted coffee beans. This method proved to be an accurate, sensitive, and less-expensive method that employs routine materials and available equipment. Although the immunoaffinity column/HPLC procedure tested showed a significantly higher percentage than the AOAC recommended method, it is recommended for use in processed coffee beans where low concentrations of OTA may be expected to be detected.     

Development of ochratoxin A during robusta (Coffea canephora) coffee cherry drying

The occurrence and formation of ochratoxin A (OTA) in Robusta coffee was studied for three consecutive seasons under tropical conditions in Thailand. Sun drying of coffee cherries consistently led to OTA formation in the pulp and parchment (husks) of the cherries. In replicated trials, dried coffee beans (green coffee) were shown to contain on average OTA concentrations that were approximately 1% of those found in husks. OTA contamination of green coffee depended on cherry maturity, with green cherries being the least, and overripe cherries the most susceptible. Defects, and in particular the inclusion of husks, are the most important source of OTA contamination. OTA contamination occurred independently of whether cherries were placed on concrete, on bamboo tables, or on the ground. The study suggests that better raw material quality, an appropriate drying and dehulling procedure combined with a reduction of green coffee defects can effectively contribute to the reduction of OTA in green coffee.     

Incidence,level, and behavior of aflatoxins during coffee bean roasting and decaffeination

Screening for aflatoxins (Afs), isolation and identification of Aspergillus flavus, and the effect of decaffeination and roasting on the level of contamination in coffee beans are studied. The percent frequency of A. flavus ranged between 4 and 80% in green coffee beans (GCB), whereas in ground roasted coffee beans (GRCB), it ranged between 1 and 71%. Aflatoxins were detected in 76.5 and 54.6% of the infected samples with averages of 4.28 and 2.85 microg/kg of GCB and GRCB, respectively. Roasting was demonstrated to lower the concentration of Afs in GCB. The Afs levels were reduced by approximately 42.2-55.9% depending on the type and temperature of roasting. The highest yields of Afs were detected in the decaffeinated green coffee beans (24.29 microg/kg) and roasted coffee beans (16.00 microg/kg). The growth of A. flavus in liquid medium containing 1 or 2% caffeine was reduced by 50%, and the level of aflatoxin in the medium was undetectable.     

Quantitation of ochratoxin A in South African wines

The natural occurrence of the carcinogenic mycotoxin ochratoxin A (OTA) in wines sold in local retail outlets in South Africa and Italy was investigated by HPLC analysis with fluorescence detection following cleanup by immunoaffinity column. All 24 local South African wines tested (15 white and 9 red) were found to contain detectable levels (>0.01 microg/L) of OTA, with a mean of 0.16 microg/L in the white wines and a mean of 0.24 microg/L in the red wines. Results were subsequently confirmed by LC-MS analysis using positive ion electrospray ionization with collision-induced dissociation of the protonated molecular ion [M + H](+) at m/z 404 and selected reaction monitoring of the resultant product ions [M + H - H(2)O - CO](+) at m/z 358 and [M + H - H(2)O](+) at m/z 386. Comparison with the fluorescence method gave a significant correlation (r = 0.87; p < 0.01). Although OTA contamination was present in all of the South African samples analyzed, levels were well below the suggested European Union limit of 0.5 microg/kg. The highest level found in a locally purchased wine was 0.39 microg/L in a blend of local and imported Spanish red wine. Of the eight Italian wines analyzed, only two red wines were contaminated above the suggested maximum level.     

Effect of roasting conditions on reduction of ochratoxin a in coffee

A commercial lot of green coffee, naturally contaminated with ochratoxin A (OTA), was roasted under various conditions, and the effects on its final OTA content were determined. Precautions were taken in sampling the coffee to cope with OTA inhomogeneity. The roasting conditions were kept within the range of commercial practice. Roasting time was varied from 2.5 to 10 min, and the roast color varied from light medium to dark. The differences in OTA reduction between the different levels of roasting times and colors did not reach statistical significance. However, for all roasting conditions, the reduction was highly significant, 69% reduction over the combined results. In total, nine studies by various authors about OTA reduction during coffee roasting are now available. Seven out of these nine reported that the relevant range of OTA reductions was between 69 and 96%. Among these seven,are all four studies that reported using naturally contaminated beans, a sampling procedure adapted to mycotoxin inhomogeneity, and roasting conditions within the range of actual practice. Three different explanations are available for this reduction: physical removal of OTA with chaff, isomerization at the C-3 position into another diastereomer, and thermal degradation with possible involvement of moisture. All three explanations may play a partial role in the OTA reduction during coffee roasting.     

Ochratoxin A on green coffee: influence of harvest and drying processing procedures

Ochratoxin A is a metabolite produced by Aspergillus and Penicillium species that is nephrotoxic and possibly carcinogenic to humans. The aim of this study was to evaluate ochratoxin A contamination in green coffee obtained by different harvesting and drying operations and from fruits of different ripening stages in order to identify hazards. The research was directed to coffees from the highland area of Rio de Janeiro state (Brazil), which is traded in the domestic market. Twenty-two out of 54 samples contained ochratoxin A at levels ranging from 0.3 to 160 microg/kg. Ochatoxin A contamination levels between different ripe stage fruits were not significant (P > 0.05). "Varri??o" coffee, consisting of fruits that fell from the tree spontaneously and stayed longer on the ground before being harvested, was the most contaminated. Eleven out of 14 samples of varri??o coffee were contaminated. Three out of 10 samples from the northwestern region of the state were positive for ochratoxin at levels ranging from 10.1 to 592 microg/kg. The contaminated samples had in common the fact that they were harvested directly from the soil.     

Natural occurrence of ochratoxin A and antioxidant activities of green and roasted coffees and corresponding byproducts

Ochratoxin A is an important mycotoxin that can enter the human food chain in cereals, wine, coffee, spices, beer, cocoa, dried fruits, and pork meats. Coffee is one of the most common beverages and, consequently, it has a potential risk factor for human health related to ochratoxin A exposure. In this study, coffee and corresponding byproducts from seven different geographic regions were investigated for ochratoxin A natural occurrence by HPLC-FLD, nutritional characterization, and antioxidant activities by spectrophotometric assay. The research focused on composition changes in coffee during the processing step "from field to cup". Costa Rica and Indian green coffees were the most contaminated samples, with 13 and 11 microg/kg, respectively, while the Ethiopian coffee was the least contaminated, with 3.8 microg/kg of ochratoxin A. The reduction of ochratoxin A contamination during the roasting step was comparable for any samples that were considered under the recommended level of 4 microg/kg. Total dietary fibers ranged from 58.7% for Vietnam and 48.6% for Ivory Coast in green coffees and ranged from 58.6% for Costa Rica to 61.2% for India in roasted coffee. Coffee silverskin byproduct obtained from Ivory Coast was the highest, with 69.2 and 64.2% of insoluble dietary fibers, respectively.     

Assay of ochratoxin A in wine and beer by high-pressure liquid chromatography photodiode array and gas chromatography mass selective detection.

To routinely assay the concentrations of ochratoxin A (OTA) in wines and beers, two new methods were developed and evaluated. The first utilized solid-phase extraction on a C(18) cartridge to achieve a 100-fold sample concentration followed by high-performance liquid chromatography on a C(18) column with gradient elution and quantitation at 333 nm by means of a photodiode array detector. Positive confirmation can be carried out by purity and match-factor analysis as well as peak shift following esterification with BF(3). Total run time is 28 min. The limits of detection (LOD) and quantitation (LOQ) are 0.05 and 0.10 microg/L, respectively. Recovery and imprecision ranged from 83 to 94% and from 4.0 to 8.9%, respectively. With a throughput of 35 assays per working day, this method is ideal for routine OTA analysis. It was used to survey the concentrations of OTA in 942 wines (2 of which gave values between 0.1 and 0.2 microg/L) and 107 beers (2 of which gave values between 0.05 and 0.1 microg/L). OTA was detected more frequently in red than white wines, with the highest incidence in red wines from Spain and Argentina. There was no association between OTA and country of origin or beverage type among the beers analyzed. The second method utilized gas chromatography with mass selective detection monitoring eight specific ions, preceded by extraction in dichloromethane and derivatization with bis[trimethylsilyl]trifluoroacetamide. LOD and LOQ were 0.1 and 2 microg/L, respectively; recovery and imprecision were 69-75 and 9.0-11.1%, respectively. The method is not suitable for routine quantitation but is potentially useful as a confirmatory tool for samples with OTA > or =0.1 microg/L.     

Analysis of ochratoxin A in foods consumed by inhabitants from an area with balkan endemic nephropathy: a 1 month follow-up study

In the 1950s, a series of publications from Bulgaria, Yugoslavia, and Romania locally described a kidney disease called Balkan Endemic Nephropathy (BEN). In Bulgaria, the exposure of populations to ochratoxin A (OTA) was supported by analysis of individual food items demonstrating a higher prevalence and higher levels of OTA in food from the high-incidence areas of BEN. In this work, food consumption from a series of individuals from two villages of the BEN area during 1 month was followed using the duplicate diet method. Meals consumed by volunteers from both villages showed uneven OTA contents, spreading from below the limit of quantification (<0.07 microg/kg) to 2.6 microg/kg. The average weekly intake of OTA varies from 1.86 to 92.7 ng/kg of body weight. Some of these levels approach the provisional tolerable weekly intake (PTWI) established by the JECFA at 100 ng/kg of body weight. These results confirm previous studies performed in the same area and demonstrate the high exposure of this population to OTA, thus strengthening the hypothesis of the involvement of this mycotoxin in BEN etiology.     

Liquid chromatographic determination of ochratoxin A in coffee beans and coffee products

A liquid chromatographic method for the determination of ochratoxin A in coffee beans (green and roast), instant coffee, and coffee drink is described. The sample is subjected to extraction with methanol-1% aqueous sodium bicarbonate (1 + 1) and C18 cartridge cleanup. The extract is chromatographed on a Nucleosil 5C18 column with a mobile solvent of acetonitrile-water-0.2M phosphate buffer pH 7.5 (50 + 47 + 3) containing 3 mM cetyltrimethylammonium bromide as an ion-pair reagent. Ochratoxin A is detected with a fluorometer (excitation 365 nm, emission 450 nm). The sensitivity was increased 20-fold by using ion-pair resolution. The detection limits corresponded to 2 micrograms/kg for coffee beans, 5 micrograms/kg for instant coffee, and 0.2 microgram/kg for coffee drink. The recoveries from coffee products were generally better than 80.7% and the relative standard deviations were 3.43-5.93%. The peak coinciding with ochratoxin A can be confirmed by treatment using alcohol (methanol, ethanol, or n-propanol) and H2SO4.     

Ochratoxin a contents in wine: comparison of organically and conventionally produced products

Ochratoxin A (OTA) content was determined in 44 organically and conventionally produced wines originating from different geographical regions. Wine samples were extracted using a series of C18 and mixed-bed solid-phase cartridges and analyzed by HPLC with fluorescence detection. The identity of the mycotoxin was confirmed using liquid chromatography-tandem mass spectrometry. Recoveries were in excess of 90%, intraday precisions were better than 6%, and the interday variation was 15%. Limit of detection was 0.05 microg/L (HPLC). All sampled wines contained OTA below the level permitted by the European Union of 2 microg/L, ranging from not detectable (nd) to 0.75 microg/L for red wines (n = 26), from nd to 0.092 microg/L for rosé wines (n = 2), and from nd to 0.22 microg/L for white wines (n = 16). The concentration of OTA in organically produced wines (nd to 0.72 microg/L, median 0.092 microg/L, n = 19) was not significantly different from that in conventional products (nd to 0.75 microg/L, median 0.066 microg/L, n = 25) as assessed by a Mann-Whitney statistical test (p = 0.54).     

Development and validation of an optical SPR biosensor assay for tylosin residues in honey

In recent years there has been an increase in the use of tylosin in apiculture as bacterial brood diseases become resistant to oxytetracycline. Confirmatory mass spectrometry based methods have been developed but up until now there has been no complementary screening method available capable of sub 10 microg kg(-1) detection limits. In this paper the development and validation of a screening method using optical biosensor technology is presented. The honey was first dissolved in a phosphate buffer and following solid-phase extraction (SPE) cleanup was analyzed using a Biacore Q instrument. Using the criteria specified in European Commission Decision 2002/657/EC for qualitative screening methods, the detection capability (CCbeta) of the method was determined to be 2.5 microg kg(-)(1). Honey samples containing trace residue levels of tylosin were analyzed by both the biosensor screening method and a LC-MS/MS confirmatory procedure; the results were in good agreement.     

Screening and quantitation of ochratoxin A in corn, peanuts, beans, rice, and cassava

To answer the need for simple, economical, rapid methods for mycotoxins, a procedure for screening and quantitation of ochratoxin A was developed. A methanol-aqueous KCl extraction is used, followed by cleanup with clarifying agents and partition into chloroform. Part of the chloroform extract is used for screening and the other part for quantitation by thin layer chromatography (TLC). The screening procedure takes 40 min, using a silica gel/aluminum oxide minicolumn developed for this purpose. The limits of detection are 80 and 10 micrograms/kg, respectively, for minicolumn screening and TLC quantitation. Ammonium sulfate is efficient in cleaning samples of corn and cassava; cupric sulfate is better with peanuts, beans, and rice. Tests were conducted on triplicate spiked samples of yellow corn meal, raw peanuts, dried black beans, polished rice, and cassava flour at different levels (400, 200, 80, 40, and 10 micrograms/kg). Recoveries ranged from 86 to 160% and the coefficients of variation ranged from 0 to 26%.     

Combination of TLC and HPLC-MS/MS methods. Approach to a rational quality control of Chinese star anise

In this study, a methodological approach for an effective and reliable quality control of Chinese star anise (Illicium verum Hook. F.) is developed and validated. A combined method of TLC and HPLC-MS/MS was used for differentiation of various Illicium species, especially Chinese and Japanese star anise. Species can be distinguished by their TLC flavonoid pattern. A sensitive and selective HPLC/ESI-MS/MS method was developed for the detection and quantification of lower admixtures of I. anisatum and of further toxic Illicium species at a low concentration range using the sesquiterpene lactone anisatin as a marker. The proposed assay includes a solid-phase extraction cleanup procedure with a high recovery (>90%). Chromatographic separation of anisatin was carried out on a C18 column, followed by MS detection using ESI in negative mode. The precursor/product ion transitions m/z 327 --> 127 (quantifier) and m/z 327 --> 297 (qualifier) were monitored. Statistical evaluation of this multireaction-monitoring procedure reveals good linearity and intra- and interday precision. The limits of detection and quantification are 1.2 and 3.9 microg/kg, respectively.     

Development of a chemiluminescent ELISA for determining chloramphenicol in chicken muscle

An indirect competitive enzyme-linked immunosorbent assay (ELISA) with chemiluminescent (CL) detection for chloramphenicol (CAP) in chicken muscle was developed. CAP-specific polyclonal antibody was raised in rabbit with a CAP-succinate derivative conjugated with bovine serum albumin. Luminol solution was used as the substrate of horseradish peroxidase. The detection limit was 6 ng/L. The CL-ELISA was 10 times more sensitive compared to the colorimetric-ELISA. When CAP was spiked in chicken muscle at levels of 0.05-5 microg/kg, recoveries ranged from 97 to 118% with coefficients of variation of 6-22%. In an actual residue study, the results obtained by CL-ELISA correlated well with those obtained by gas chromatography with microcell electron capture detector. The residue levels of CAP in treated chicken decreased with time and dropped rapidly after the first 6 h from around 50 to 10 microg/kg. After 3 days, CAP was not detected in chicken muscle. The developed method is therefore suitable for screening of CAP in chicken muscle samples.     

Investigation of sample treatment steps for the analysis of polycyclic aromatic hydrocarbons in ground coffee

Sample treatment procedures were tested for the determination of polycyclic aromatic hydrocarbons (PAHs) in ground coffee. Pressurized liquid extraction (PLE), under different conditions, was combined with several cleanup methods, namely in situ purification, C18-silica solid-phase extraction (SPE), silica SPE, acid digestion, and alkaline saponification. Soxhlet extraction and direct alkaline saponification were also tested. Best results were obtained using PLE with hexane/acetone 50:50 (v/v) under 150 degrees C. Alkaline saponification followed by cyclohexane extraction and silica SPE was required to eliminate interferent compounds. Finally, 11 PAHs could be quantified in ground coffee with limits of detection in the range of 0.11-0.18 microg kg(-1). Application to ground Arabica coffee lots from Colombia revealed the presence of several PAHs, giving an overall toxicity equivalence in the range of 0.16-0.87 microg kg(-1). PAH identification was performed using both high-performance liquid chromatography-diode array detection and gas chromatography coupled to mass spectrometry.