Detection of dehydroepiandrosterone and androsterone in a traditional Chinese herbal product

Dehydroepiandrosterone (DHEA) and androsterone (ADT) were detected in a traditional Chinese herbal product by gas chromatography-mass spectrometry (GC-MS) and liquid chromatography tandem mass spectrometry (LC-MS/MS). DHEA and ADT were tentatively identified by comparing their electron ionization (EI) mass spectra with those in the GC-MS Wiley database. A multiple reaction monitoring (MRM) scan was performed in LC-MS/MS to confirm the presence of the DHEA and ADT in the herbal product extract. Both the [M + H]⁺ and the [M + NH₄]⁺ of DHEA and ADT were selected as the precursor ions. DHEA was detected with ion transitions m/z 306.4 → 271.2, 306.4 → 253.3, 289.2 → 270.9, 289.3 → 253.1 while ADT was detected with ion transitions m/z 308.5 → 273.6, 308.5 → 255.3, 291.5 → 273.5, 291.5 → 255.2, which confirmed the presence of the two steroid hormones in the herbal product. Limits of detection (LODs) of 0.2 µg ml^-1 for DHEA and 0.3 µg ml^-1 for ADT were found in methanolic standard solutions when [M + NH₄]⁺ of DHEA and ADT were selected as the precursor ions, which allowed the detection of DHEA and ADT at trace level without time-consuming derivatization.     

An intercomparison study of the determination of glyphosate, chlormequat and mepiquat residues in wheat

An intercomparison study of the determinations of glyphosate, chlormequat and mepiquat residues in cereals was performed. Four samples comprising one blank, two incurred and one spiked sample were sent to six participating laboratories. For glyphosate, two laboratories reported considerably lower results than the other four. One of the two laboratories with low results also reported low recoveries. The results of a sample spiked with 0.80 mg kg^-1 glyphosate and an incurred sample, ranged from 0.23-0.87 mg kg^-1 and 0.11-0.25 mg kg^-1 respectively. The strong correlation between the two samples (r² = 0.95) indicates a systematic between-laboratory variation. Several different principles were used for the analysis of glyphosate using different clean-up techniques and GC/MS, HPLC-fluorescence or LC/MS for detection. The results of the chlormequat residues showed more consistency. All but one laboratory obtained comparable results. However the correlation between the results for the sample spiked with 0.38 mg kg^-1 (range: 0.26-0.65 mg kg^-1) and the incurred samples (range: 0.19-0.45 and 0.15-0.23 mg kg^-1, respectively) again showed a strong correlation (r² = 0.99 and 0.88) indicating a systematic component. For mepiquat, results above the limit of quantification were only reported for the spiked sample. The results ranged from 0.29-0.92 mg kg^-1 (spiked concentration = 0.38 mg kg^-1). Three laboratories had results that deviated less than 25% from the fortified concentration. Two laboratories reported results 38% and 141% above the fortified concentration, respectively.     

Residues of β-lactam antibiotics in bovine milk: confirmatory analysis by liquid chromatography tandem mass spectrometry after microbial assay screening

A procedure for the simultaneous determination of the residues of seven β-lactam antibiotics (penicillin G, ampicillin, oxacillin, amoxicillin, dicloxacillin, cephalexin, cephapirin) in bovine raw milk using tandem liquid chromatography mass spectrometry (LC-MS/MS) is described. The antibiotics were extracted by an acetic acid solution after centrifugation and filtration. The β-lactams were separated using reversed-phase liquid chromatography. A triple quadrupole mass spectrometer was used in positive-ion mode as a detector via a Turbo Ionspray interface for electrospray ionization (ESI). The limits of detection and quantitation of the method were below the legal tolerances, except for ampicillin. The method was used to confirm 53 samples found positive by a microbial method (Delvotest SP®) at the Istituto Zooprofilattico Sperimentale per la Lombardia e l'Emilia Romagna of Brescia during 2001. Penicillin G was found in 26 samples at concentrations ranging from less than 4 to 6240 ±550 μg l^-1. Amoxicillin was found in three samples at concentrations ranging from 8.5 ±0.1 to 53.7 ±2.3 μg l^-1. Cephapirin was found in two samples at 5.7 ±0.1 and 6.4 ±0.3 μg l^-1.     

Determination of ochratoxin A in grapes of Greek origin by immunoaffinity and high-performance liquid chromatography

A simple analytical method for ochratoxin A (OTA) determination in grapes is described, using aqueous methanolic extraction, an immunoaffinity column clean-up step and high-performance liquid chromatography with fluorescence detection. Mean recovery was 94% (RSD = 4.0%) with a detection limit of 0.4 ng g^-1 and quantification limit of 1.20 ng g^-1. Repeatability (r) and reproducibility (R) were 1.17 and 1.34, respectively. OTA determinations were applied to 50 grape samples (23 different varieties) originating from representative regions of Greece. Results showed the presence of OTA in 86% of samples tested (n = 50). Traces were found in 56% of samples but OTA was not detectable in 14% of samples. Traces were also found in 4% of red, organically grown samples. The most contaminated were three samples of red grapes, two from Central Greece (2.69 and 1.41 ng g^-1), both table and wine-making grapes. The third sample (1.46 ng g^-1), originating from the island of Samos, was used only in wine-making. Mean (1.06 ng g^-1) and median (0.76 ng g^-1) OTA concentrations in red grapes were slightly higher compared to the mean (0.82 ng g^-1) and median (0.65 ng g^-1) concentrations in white grape samples. The study shows that the potential risk for a person of 60 kg ranged from 0.9 to 9 ng kg^-1 bw day^-1 and is dependent on the quantity of grapes consumed daily.     

Occurrence of fumonisins B1 and B2 in Portuguese maize and maize-based foods intended for human consumption

Fumonisin B₁ (FB₁) and fumonisin B₂ (FB₂) are mycotoxins mainly produced by Fusarium verticillioides and Fusarium proliferatum, which are field pathogens of maize. A survey was conducted on the incidences of FB₁ and FB₂ in both maize and derived products purchased in Portugal. The analytical method involved extraction with methanol-water, clean-up by immunoaffinity column and derivatization with naphthalene-2,3-dicarboxaldehyde. Determination was carried out by high-performance liquid chromatography (HPLC) with spectrofluorimetric detection, with liquid chromatography/mass spectrometry (LC/MS) confirmation. The presence of FB₁ and FB₂ was determined in 67 samples of maize and maize-based foods, such as flour, semolina, starch, sweet maize, cornflakes and other breakfast cereals, and snacks collected in 2005. FBs were found in 15 samples at concentrations ranging from 113 to 2026 µg kg^-1. Two of the samples showed higher contamination levels than the limits established by the European Commission Regulation. None of the samples contained levels of fumonisins that would lead to an exposure exceeding the tolerable daily intake (TDI).     

Mycotoxin occurrence and Aspergillus flavus soil propagules in a corn and cotton glyphosate-resistant cropping systems

The effects of cotton-corn rotation and glyphosate use on levels of soil-borne Aspergillus flavus, aflatoxin and fumonisin contamination in corn and cotton seed were determined during 2002-2005 in Stoneville, Mississippi (USA). There were four rotation systems (continuous cotton, continuous corn, cotton-corn and corn-cotton) for both glyphosate-resistant (GR) and non-GR cultivars-herbicide system arranged in a randomized complete block design with four replications. Aspergillus flavus populations in surface (5-cm depth) soil, sampled before planting (March/April), mid-season (June) and after harvest (September), ranged from 1.47 to 2.99 log (10) cfu g^-1 soil in the four rotation systems. Propagules of A. flavus were higher in the continuous corn system compared to the continuous cotton system on three sample dates, and cotton rotated with corn decreased A. flavus propagules in three of nine sample dates. Propagules of A. flavus were significantly greater in plots with GR cultivars compared to non-GR cultivars in three samples. In cotton seed, aflatoxin and fumonisin levels were similar (≤4 µg kg^-1 and non-detectable, respectively) regardless of rotation and glyphosate. In corn grain, aflatoxin was above the regulatory level (≥20 µg kg^-1) only in GR cultivar in 2004 and 2005. Fumonisin was higher in non-GR cultivar (4 mg kg^-1) regardless of rotation in 2004; however, in 2002, 2003 and 2005, aflatoxin and fumonisin levels were similar regardless of rotation and glyphosate. These results indicate the potential for increased aflatoxin and fumonisin levels (1 of 4 years) in corn; however, climatic conditions encountered during this study did not allow for mycotoxin production. In laboratory incubation studies, fairly high concentrations of glyphosate were required to inhibit A. flavus growth; however no short-term effect of soil treatment with glyphosate on A. flavus populations were observed. These data suggest that altered populations of A. flavus or higher aflatoxin concentrations in corn grain were due to indirect effects of the GR cropping system.     

Survey of ochratoxin A and deoxynivalenol in stored grains from the 1999 harvest in the UK

Three hundred and twenty samples from the 1999 UK harvest comprising wheat (201 samples), barley (106) and oats (13) were analysed for ochratoxin A and deoxynivalenol. A small number of organic samples was also obtained. Samples were collected from farms, central stores, mills, maltings and ports from across the UK from February to April 2000. Ochratoxin A and deoxynivalenol analysis was by affinity column clean up and high-performance liquid chromatography with fluorescence and ultraviolet light detection, respectively, with limits of detection of 0.2 and 20 μg kg^-1. The survey found ochratoxin A at below 5 μg kg^-1 in 97% of the samples indicating satisfactory storage conditions. The remaining 3% of the samples contained ochratoxin A at levels between 5.2 and 231 μg kg^-1, but none of these samples was intended for human consumption. Deoxynivalenol was detected in 88% of all samples, with 83% below 100 μg kg^-1; the maximum level was 600 μg kg^-1. Twenty samples containing deoxynivalenol at or above 150 μg kg^-1 by high-performance liquid chromatography were all confirmed by gas chromatography/mass spectrometry. Nivalenol was also detected by gas chromatography/mass spectrometry at levels of 50 μg kg^-1 or higher in 18 of 20 samples where deoxynivalenol was confirmed.     

Five-year survey of ochratoxin A in processed sultanas from Turkey

The results of surveillance for ochratoxin A (OTA) in 1885 samples of sultanas taken during five crop years between 1999 and 2003 are reported. The analytical method was based on extraction with methanol + sodium bicarbonate and clean-up by immunoaffinity column chromatography followed by high-performance liquid chromatography with fluorescence detection. The limit of detection for OTA was 0.3 µg kg^-1. The results show that 9.3% of the samples contained no detectable levels of OTA, whereas 0.6% had concentrations exceeding 10 µg kg^-1; the remaining 90.3% had levels within the range 0.3-10 µg kg^-1. The overall mean OTA concentration in the total number of 1885 samples taken was 1.36 ± 2.91 µg kg^-1; the overall median was calculated as 0.90 µg kg^-1.     

Determination of ochratoxin A in virgin olive oils of Greek origin by immunoaffinity column clean-up and high-performance liquid chromatography

An improved specific analytical method for ochratoxin A (OTA) determination in olive oil is described, using a methanolic-aqueous extraction, an immunoaffinity column clean up step and high-pressure liquid chromatography with fluorescence detection. The mean recovery was found at 108% (relative standard deviation, RSD = 4.7%) and the detection limit (DL) was estimated at 4.6 ng kg^-1. Along with OTA, aflatoxin B₁ (AFB₁) was determined using the same extract. The recovery factor was 84.8% (RSD = 17.8%) and the DL was 56 ng kg^-1 olive oil. Both determinations were applied in 50 samples of olive oil originated from representative regions of Greece. Results revealed the presence of OTA in 88% of samples tested (n = 44, mean 267 ng kg^-1). Among them, 10 were contaminated with more than 500 ng kg^-1 (median 568 ng kg^-1), 10 with 200-500 ng kg^-1 (median 260 ng kg^-1), 15 with 100-200 ng kg^-1 (median 140 ng kg^-1), nine with DL-100 (median 60 ng kg^-1) and in six samples, OTA was not detectable. Interestingly, most contaminated samples were from Southern Greece. Results of AFB₁ determination showed the presence of aflatoxin B₁ (60 ng kg^-1) in only one olive oil sample also from Southern Greece. The levels of OTA found in Greek olive oil were relatively low as compared with other commodities such as cereals or wine reported in the literature.     

Ochratoxin A in dried vine fruits on the Canadian retail market

Between 1998 and 2000, 151 samples of raisins and sultanas and two samples of currants were collected from retail outlets across Canada and analysed for ochratoxin A. Samples were extracted with methanol-sodium bicarbonate, and the extracts were cleaned-up by immunoaffinity column chromatography. Ochratoxin A was quantified by liquid chromatography with fluorescence detection. The minimum quantifiable level was 0.1 ng g^-1. Ochratoxin A was present, above the minimum quantifiable level, in 67 (79%) of 85 samples of raisins, in 39 (59%) of 66 samples of sultanas, and in both samples of currants. The overall mean level of ochratoxin A was 1.8 ng g^-1 in both the raisins and sultanas, and 2.8 ng g^-1 in the currants.     

Quantitative analysis of Fusarium mycotoxins in maize using accelerated solvent extraction before liquid chromatography/atmospheric pressure chemical ionization tandem mass spectrometry

A method for the simultaneous quantitative determination of deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone (ZEN) in maize by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry (LC-APCIMS/MS), using stable isotopically labelled and structural analogues internal standards, is described. The procedure involves accelerated solvent extraction followed by two solid-phase clean-up steps on strong anion exchange resin and a Mycosep® column. Typical recoveries were calculated by spiking blank maize at three different concentrations for deoxynivalenol (200, 400 and 1000 μg kg^-1) at 70%, for fumonisin B1 (100, 200 and 1000 μg kg^-1) at 90%, and for zearalenone (50, 100 and 200 μg kg^-1) at 40%. LC-APCIMS/MS analyses were realized in collision-induced dissociation on an ion-trap instrument to provide a high degree of selectivity and sensitivity. Extraction of ions from two transition reactions, monitored by LC-APCIMS/MS for each analyte, enabled a limit of detection for DON, FB1 and ZEN at, respectively, 10, 20 and 3 μg kg^-1, and a limit of quantification at, respectively, 50, 50 and 10 μg kg^-1. The robustness of the method was also evaluated with the analysis of wheat samples.     

Occurrence of aflatoxin M1 in domestic milk in Japan during the winter season

A total of 208 samples of commercial pasteurized milk gathered from retail outlets across Japan during the winter season were analysed for aflatoxin M₁ (AFM₁). Japan was divided into 11 regions from north to south, and nine to 45 milk samples from each region were randomly purchased between December 2001 and February 2002. Each milk sample was cleaned up by an immunoaffinity column, and AFM₁ was quantified by liquid chromatography with fluorescence detection in four independent laboratories. The limit of detection of the method was 0.001 μg kg^-1. The identity of the putative AFM₁ in milk sample was confirmed by the formation of AFM₁ hemi-acetal with trifluoroacetic acid. Based on the results obtained with spiked samples (0.05 μg AFM₁ kg^-1), the mean recovery was 91.4%, the relative standard deviation for repeatability was 4.6%, and the relative standard deviation for reproducibility was 8.0% among four independent laboratories. AFM₁ was detected in 207 (99.5%) of 208 milk samples at 0.001-0.029 μg kg^-1, with a mean of 0.009 μg kg^-1 and a 90th percentile of 0.014 μg kg^-1. No significant difference of the level of AFM₁ contamination was observed among the regions.     

Clostridium perfringens and its toxins in minced meat from Kars, Turkey

The aim of this study was to determine the prevalence of Clostridium perfringens and its toxins in minced meat. A total of 96 minced meat samples were collected from local markets (16) and small butcher's shops (80) in Kars (Turkey). Samples were analysed for the presence of C. perfringens and its toxins using a commercially available ELISA kit. A total of 31 (32%) Clostridium spp. strains were isolated and 17 (55%) of these isolates were identified as C. perfringens. Four (25%) of the samples from local markets and 27 (34%) from small butcher's shops were contaminated with Clostridium spp. Furthermore, C. perfringens was isolated from two (12%) and 15 (19%) samples from local markets and small butcher's shops, respectively. Mean counts of Clostridium spp. were 2.2 ± 0.83 × 10² CFU g^-1 for local markets and 4.35 ± 8.53 × 10² CFU g^-1 for small butcher's shops; mean counts for C. porringers were 2.75 ± 0.21 × 10² and 6.82 ± 10.96 × 10² CFU g^-1 from markets and butcher's shops, respectively. The number of samples contaminated with both Clostridium spp. and C. perfringens was higher in small butcher's shops than in local markets. Moreover, higher numbers of Clostridium spp. and C. perfringens were isolated in samples from small butcher's shops than from local markets. A total of 13 (13%) samples were also positive for toxins produced by the organism, as detected by ELISA. Eleven samples from small butcher's shops and two samples from local markets were positive for the C. perfringens toxins tested. Moreover, two (12%), one (1%), four (4%) and two (2%) samples were contaminated with C. perfringens types A, B, C and D, respectively. In conclusion, some meat samples collected from local markets and small butcher's shops contained C. perfringens and its toxins and, therefore, present a potential risk of food poisoning.     

Analysis and monitoring of chloramphenicol residues in food of animal origin in Slovenia from 1991 to 2000

To prevent the illegal use of chloramphenicol (CAP), regulatory control of its residues in food of animal origin is essential. In Slovenia, the monitoring of CAP residues for statutory purpose started in 1991. The results of a 10-year period are presented. CAP residues were determined by capillary gas chromatography (GC) with electron capture detection (ECD) using meta-CAP as an internal standard (ISTD). Before chromatographic determination, analytes were derivatized by silylation. Overall, CAP recovery, adjusted for ISTD, was for bovine muscle tissue and raw cow's milk (in the region of 2-10 μg kg^-1) 89 and 102%, respectively, and for whole eggs, 87% (in the region of 1-10 μg kg^-1). The use of meta-CAP improved significantly the precision of the method. The detection limit for CAP was 1 μg kg^-1, which was sufficiently sensitive for routine use. A total of 1308 random samples of Slovenian origin were analysed from 1991 to 2000, covering all parts of the country. CAP was found only in one milk sample in 1997 at a concentration of 4.6 μg kg^-1.     

Determination of 1-phenylazo-2-naphthol (Sudan I) in chilli powder and in chilli-containing food products by GPC clean-up and HPLC with LC/MS confirmation

A simple and rapid method is reported for the routine determination 1-phenylazo-2-naphthol (Sudan I) in chilli powder and in chilli-containing food products. The method involved Soxtec extraction from the products followed by high-pressure gel permeation chromatographic clean-up collecting the appropriate fraction. Analysis of this fraction was by HPLC with UV/VIS detection. The limit of detection was 7 μg kg^-1 and the limit of quantification was 13 μg kg^-1. The identity of Sudan I in food products was established by electrospray LC/MS with MS/MS confirmation. From a small survey of 30 retail samples, 11 samples of crushed chilli, Italian pasta, chilli-snack and vegetable sauce contained levels of Sudan I ranging from 24 to 5591 μg kg^-1.     

Analysis of vinylidene chloride and 1-chlorobutane in foods packaged with polyvinylidene chloride casing films by headspace gas chromatography/mass spectrometry (GC/MS)

A headspace gas chromatography/mass spectrometry method was developed for the simultaneous determination of vinylidene chloride and 1-chlorobutane in foods packaged with polyvinylidene chloride casing films. The solid foodstuff was homogenized with an equal mass of distilled water. The homogenate was incubated for 1 h at 90°C in a sealed headspace vial, and the headspace gas was then analysed by gas chromatography/mass spectrometry in selected ion-monitoring mode using a bonded porous polymer-coated capillary column. The recovery rates of vinylidene chloride and 1-chlorobutane in foodstuffs were 94.5-103.9 and 85.8-120.3%, respectively. Among 13 samples tested, vinylidene chloride was detected at 0.001-0.020 µg g^-1 in 11 foodstuffs, and 1-chlorobutane was detected at 0.004-0.040 µg g^-1 in all 13 foodstuffs. Furthermore, vinylidene chloride was detected at 0.04 µg g^-1 in one casing film, and 1-chlorobutane was detected in all casing films. The results indicate that these compounds migrated from the casing films into the foodstuffs.     

Characterization of a Non-electrophoretic Genetic Variant of β-Casein by Peptide Mapping and Mass Spectrometric Analysis

From a total of 634 Holstein milk samples, 158 were identified as homozygous β-casein A¹ according to PAGE. All the β-casein A¹ samples were isolated in pure form by anion exchange chromatography followed by tryptic hydrolysis and peptide mapping of the hydrolysates by reversed-phase HPLC. A comparison of the chromatogram revealed that the β-casein A¹ samples could be classified into two groups based on differences in elution times of fragment 114–169 due to a change in hydrophobicity caused by amino acid substitution. Mass spectrometric analysis showed that 7 β-casein samples with more hydrophobic 114–169 fragment had a higher molecular mass (difference of 16 Da) than the remaining 151 samples with less hydrophobic fragment. Amino acid composition and sequence analysis confirmed the difference in mass of peptide 114–169 by a Pro→Leu substitution at position 137. It is proposed to identify the newly found variant as β-casein G.     

A sensitive solid-phase microextraction/gas chromatography-based procedure for determining pentachlorophenol in food

A new method for the determination of pentachlorophenol (PCP) in different foods was developed using capillary gas chromatography (GC) and microwave induced-plasma atomic emission spectrometry (MIP-AED). The analyte is first derivatized and then extracted and pre-concentrated by solid-phase microextraction (SPME) in headspace (HS) mode. A clear matrix effect was found for the different samples investigated, so that standard addition was required for quantification. Detection limits of 0.03-6.0 ng g^-1 were obtained, depending on the sample analysed. The method gave recoveries of 81-109% from spiked samples. Concentration levels of PCP ranged from 0.3 to 1.5 ng g^-1 were found in honey, but no PCP was detected in other samples.     

Development and application of a simplified clean-up procedure for the determination of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in horse fat by gas chromatography-tandem mass spectrometry (GC-MS/MS)

A simplified clean-up procedure was developed in combination with gas chromatography-tandem mass spectrometry (GC-MS/MS) for the determination of polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs) in adipose tissue. Clean-up was performed by the successive application of a Mega Bond Elut® silica column and a Bond Elut® PCB column. Validation of the method was conducted according to European Union Commission Decision 2002/657/EC. In order to evaluate the applicability of the method, 44 horse fat samples were analysed. The total PCB concentration (sum of PCBs 28, 52, 101, 118, 138, 153 and 180) ranged from 5.35 to 140 ng g^-1 lipid weight. The total PBDE concentration (sum of BDEs 28, 47, 99, 100, 153, 154 and 183) ranged from below the decision limit to 6.34 ng g^-1 lipid weight.     

Levels of eight trace elements in edible mushrooms from a rural area

Eight trace elements were determined using ICP-MS in 78 fruiting body samples of 22 edible mushroom species. The mushrooms were collected from four sites in a rural area, unpolluted by human activity. Median values (dry matter) were as follows: Arsenic (As) 1.45 mg kg^-1, barium (Ba) 1.41 mg kg^-1, cobalt (Co) 0.28 mg kg^-1, copper (Cu) 47.0 mg kg^-1, rubidium (Rb) 130 mg kg^-1, silver (Ag) 2.95 mg kg^-1, thallium (Tl) 0.02 mg kg^-1 and vanadium (V) 0.25 mg kg^-1. Higher trace element accumulation was observed in samples of Macrolepiota procera, Macrolepiota rhacodes, Lycoperdon perlatum, Lycoperdon gigantea and Xerocomus chrysenteron for As and Cu, and in samples of Cantharellus cibarius and of genera Boletus and Suillus for Rb.