Determination of deoxynivalenol in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered a applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200-2000 ng/g deoxynivalenol. Average recoveries ranged from 78 to 87%. Based on results for 6 artificially contaminated samples (blind duplicates), the relative standard deviation for repeatability (RSDr) ranged from 3.1 to 14.1%, and the relative standard deviation for reproducibility (RSDR) ranged from 11.5 to 26.3%. The method showed acceptable within-laboratory and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values < 1.3.     

Determination of water (moisture) and dry matter in animal feed, grain, and forage (plant tissue) by Karl Fischer titration: collaborative study

A Karl Fischer method for determining water (dry matter) in animal feed and forages was collaboratively studied. Water was extracted from animal feed or forage material into methanol-formamide (1 + 1) directly in the Karl Fischer titration vessel by high-speed homogenization. The water was titrated at 50 degrees C with one-component Karl Fischer reagent based on imidazole. Ten blind samples were sent to 9 collaborators in the United States, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.14 to 6.99% for water or from 0.09 to 0.56% for dry matter. Among-laboratory (including within-) relative standard deviation (reproducibility) ranged from 5.35 to 10.73%, or from 0.44 to 0.77% for dry matter. The authors recommend that the method be adopted as Official First Action by AOAC INTERNATIONAL. A comparable alternative extraction procedure using boiling methanol is also recommended for Official First Action.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in peanut butter, pistachio paste, fig paste, and paprika powder: collaborative study

A collaborative study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 and total aflatoxins at European regulatory limits. The test portion is extracted with methanol-water (8 + 2) for dried figs and paprika, and with methanol-water (8 + 2) plus hexane (or cyclohexane) for peanut butter and pistachios. The sample extract is filtered, diluted with phosphate buffer saline, and applied to an immunoaffinity column. The column is washed with water and the aflatoxins are eluted with methanol. Aflatoxins are quantitated by reversed-phase LC with post-column derivatization (PCD) involving bromination. PCD is achieved with either an electrochemical cell (Kobra cell) and addition of bromide to the mobile phase or pyridinium hydrobromide perbromide. Determination is by fluorescence. Peanut butter, pistachio paste, dried fig paste, and paprika powder samples, both naturally contaminated with aflatoxins and containing added aflatoxins, were sent to 16 collaborators in 16 European countries. Test portions of samples were spiked at levels of 2.4 and 9.6 ng/g for total aflatoxins which included 1.0 and 4.0 ng/g aflatoxin B1, respectively. Recoveries for total aflatoxins ranged from 71 to 92% with corresponding recoveries for aflatoxin B1 of 82 to 109%. Based on results for spiked samples (blind duplicates at 2 levels) as well as naturally contaminated samples (blind duplicates at 4 levels, including blank), the relative standard deviation for repeatability ranged from 4.6 to 23.3% for total aflatoxins and from 3.1 to 20.0% for aflatoxin B1. The relative standard deviation for reproducibility ranged from 14.1 to 34.2% for total aflatoxins, and from 9.1 to 32.2% for aflatoxin B1. The method showed acceptable within-laboratory and between-laboratory precision for all 4 matrixes, as evidenced by HORRAT values <1, at the low levels of determination for both total aflatoxins and aflatoxin B1.     

Liquid chromatographic determination of deoxynivalenol in baby food and animal feed: interlaboratory study

An interlaboratory study was conducted for the determination of deoxynivalenol in baby food and animal feed by high-performance liquid chromatography with UV detection. The study included 14 participants representing a cross section of industry, official food control, and research facilities. Mean recoveries reported ranged from 89% (at 120 microg/kg) to 85% (at 240 microg/kg) for baby food and from 100% (at 200 microg/kg) to 93% (at 400 microg/kg) for animal feed. On the basis of the results for spiked samples (blind duplicates at 2 levels), as well as those for naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in analyses of baby food ranged from 6.4 to 14.0% and in analyses of animal feed, from 6.1 to 16.5%. The relative standard deviation for reproducibility (RSDR) in analyses of baby food ranged from 9.4 to 19.5% and in analyses of animal feed, from 10.5 to 25.2%. The HorRat values ranged from 0.4 to 1.0 and from 0.7 to 1.3, for baby food and animal feed, respectively. The method showed acceptable performance for within-laboratory and between-laboratory precision for each matrix, as required by European legislation.     

Determination of zearalenone in cereal grains, animal feed, and feed ingredients using immunoaffinity column chromatography and liquid chromatography: interlaboratory study

A method using immunoaffinity column chromatography (IAC) and liquid chromatography (LC) for determination of zearalenone in cereal grains, animal feed, and feed ingredients was collaboratively studied. The test portion is extracted by shaking with acetonitrile-water (90 + 10, v/v) and sodium chloride. The extract is diluted and applied to an immunoaffinity column, the column is washed with water or phosphate-buffered saline or methanol-water (30 + 70, v/v), and zearalenone is eluted with methanol. The eluate is evaporated, the residue is dissolved in mobile phase and analyzed by reversed-phase LC with fluorescence detection. The presence of zearalenone can be confirmed using an alternate excitation wavelength or diode array detection. Twenty samples were sent to 13 collaborators (8 in Europe, 2 in the United States, one in Japan, one in Uruguay, and one in Canada). Eighteen samples of naturally contaminated corn, barley, wheat, dried distillers grains, swine feed, and dairy feed were analyzed as blind duplicates, along with blank corn and wheat samples. The analyses were done in 2 sample sets with inclusion of a spiked wheat control sample (0.1 mg/kg) in each set. Spiked samples recoveries were 89-116%, and for the 18 naturally contaminated samples, RSDr values (within-laboratory repeatability) ranged from 6.67 to 12.1%, RSDR values (among-laboratory reproducibility) ranged from 12.5 to 19.7%, and HorRat values ranged from 0.61 to 0.90.     

Determination of decoquinate in animal feeds by liquid chromatography: collaborative study

The performance characteristics of a liquid chromatographic (LC) method for the analysis of decoquinate (DEC) in supplements, premixes, and complete animal feeds at medicating and trace levels were collaboratively studied. DEC is extracted from ground feed samples with 1% calcium chloride-methanol solution using mechanical agitation for 90 min. After centrifugation for 5 min and dilution (if necessary), an aliquot of the extract is diluted with water. The diluted extracts are filtered and analyzed by reversed-phase LC with fluorescence detection. Suspect positive trace-level samples are confirmed by using an alternate excitation wavelength. Fourteen test samples of medicated feeds, supplement, and medicated premix, along with 8 test samples for trace-level analysis, were sent to 13 collaborators (one in Canada, 4 in Europe, and 8 in the United States). Test samples were analyzed as blind duplicates. Acceptable results were received from 12 laboratories for the medicated test samples and from 13 laboratories for the trace-level samples. Repeatability relative standard deviation estimates ranged from 1.3 to 5.6%. Reproducibility relative standard deviations estimates ranged from 2.8 to 6.1%, and HorRat values ranged from 0.22 to 0.74.     

Crude fat, diethyl ether extraction, in feed, cereal grain, and forage (Randall/Soxtec/submersion method): collaborative study

A method for determining crude fat in animal feed, cereal grain, and forage (plant tissue) was collaboratively studied. Crude fat was extracted from the animal feed, cereal grain, or forage material with diethyl ether by the Randall method, also called the Soxtec method or the submersion method. The proposed submersion method considerably decreases the extraction time required to complete a batch of samples. The increase in throughput is very desirable in the quest for faster turnaround times and the greater efficiency in the use of labor. In addition, this method provides for reclamation of the solvent as a step of the method. The submersion method for fat extraction was previously studied for meat and meat products and was accepted as AOAC Official Method 991.36. Fourteen blind samples were sent to 12 collaborators in the United States, Sweden, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.09 to 9.26% for crude fat. Among-laboratory (including within) relative standard deviation (reproducibility) ranged from 1.0 to 21.0%. The method is recommended for Official First Action.     

Determination of sulfamethazine in swine and cattle feed by reversed-phase liquid chromatography with post-column derivatization: pre-collaborative study

In-house validation of a liquid chromatographic method for determination of sulfamethazine in swine and cattle feed was performed to verify that the method was ready for collaborative study under AOAC INTERNATIONAL guidelines. In this method, sulfamerazine is added during the extraction procedure and is used as an internal standard to correct for variable recovery of sulfamethazine from a variety of swine and cattle feed matrixes. The determinative step involves the use of post-column derivatization with dimethylaminobenzaldehyde which reacts with the primary amine group on the sulfonamides. Detection is at 450 nm, a wavelength at which most co-extracted matrix materials and other feed additives do not absorb light. The results indicate that the method recovery, precision, and ruggedness meet normal criteria to be ready for a collaborative study. Fortification experiments over a range of sulfamethazine concentrations from 0.006 to 0.26% showed an overall recovery relative to the internal standard of 100 +/- 2%. These studies include both swine and cattle feed matrixes. The mean recovery in the analysis of 3 beef cattle experimental feeds was 98.9%. The method results agreed with the AOAC INTERNATIONAL Official Method for colorimetric analysis of swine feed. Method precision was excellent during in-house validation studies, with coefficients of variation (CVs) ranging from about 0.5 to 3%. The method ruggedness was verified with an overall CV of 3.5%.     

Immunoaffinity column cleanup with liquid chromatography using post-column bromination for determination of aflatoxins in hazelnut paste: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for determination of aflatoxin B1 and total aflatoxins in hazelnut paste at European regulatory limits. The test portion was extracted with methanol-water (6 + 4). The extract was filtered, diluted with phosphate-buffered saline (PBS) solution to a specified solvent concentration, and applied to an immunoaffinity column containing antibodies specific to aflatoxins. The aflatoxins were removed from the immunoaffinity column with methanol, and then quantified by reversed-phase LC with post-column derivatization (PCD) involving bromination. The PCD was achieved with electrochemically generated bromine (Kobra Cell) followed by fluorescence detection (except for one participant who used pyridinum hydrobromide perbromide for bromination). Hazelnut paste, both naturally contaminated with aflatoxins and blank (<0.1 ng/g) for spiking by participants with aflatoxins, was sent to 14 collaborators in Belgium, The Netherlands, Spain, Turkey, the United Kingdom, and the United States. Test portions were spiked at levels of 4.0 and 10.0 ng/g for total aflatoxins by participants using supplied total aflatoxins standards. Recoveries for total aflatoxins and aflatoxin B1 averaged from 86 to 89%. Based on results for naturally contaminated samples (blind duplicates at 3 levels ranging from 4.0 to 11.8 ng/g total aflatoxins), the relative standard deviation for repeatability (RSDr) ranged from 2.3 to 3.4% for total aflatoxins and from 2.2 to 3.2% for aflatoxin B1. The relative standard deviation for reproducibility (RSD(R)) ranged from 6.1 to 7.0% for total aflatoxins and from 7.3 to 7.8% for aflatoxin B1. The method showed exceptionally good within-laboratory and between-laboratory precision for hazelnut paste, as evidenced by HORRAT values, which in all cases were significantly below target levels, the low levels of determination for both aflatoxin B1 and total aflatoxins.     

Crude fat, hexanes extraction, in feed, cereal grain, and forage (Randall/Soxtec/submersion method): collaborative study

A method for determining crude fat in animal feed, cereal grain, and forage (plant tissue) was collaboratively studied. Crude fat was extracted from the animal feed, cereal grain, or forage material with hexanes by the Randall method, also called the Soxtec method or the submersion method. The use of hexanes provides for an alternative to diethyl ether for fat extractions. The proposed submersion method considerably decreases the extraction time required to complete a batch of samples compared to Soxhlet. The increase in throughput is very desirable in the quest for faster turnaround times and the greater efficiency in the use of labor. In addition, this method provides for reclamation of the solvent as a step of the method. The submersion method for fat extraction was previously studied for meat and meat products and was accepted as AOAC Official Method 991.36. Fourteen blind samples were sent to 14 collaborators in the United States, Sweden, Canada, and Germany. The within-laboratory relative standard deviation (repeatability) ranged from 1.23 to 5.80% for crude fat. Among-laboratory (including within) relative standard deviation (reproducibility) ranged from 1.88 to 14.1%. The method is recommended for Official First Action.     

Determination of chloramphenicol in royal jelly by liquid chromatography/tandem mass spectrometry

A liquid chromatographic/tandem mass spectrometric method was developed and validated for the determination of chloramphenicol (CAP) in royal jelly. Royal jelly samples were first denatured with lead acetate solution, and the CAP was extracted with solid-phase extraction before separation by liquid chromatography. A triple-quadrupole mass spectrometer operated in the negative electrospray ionization and selected-reaction monitoring mode was used for the detection of CAP. For method validation, royal jelly samples were fortified at CAP levels between 0.1 and 10.0 microg/kg; at these levels, recovery values (internal standard-corrected) ranged from 93.3 to 105.0%, and the within-laboratory reproducibility (relative standard deviation) was < or = 9.1%. The decision limit was 0.07 microg/kg, and the detection capability was 0.1 microg/kg.     

Liquid chromatographic method for the quantification of zearalenone in baby food and animal feed: interlaboratory study

An interlaboratory trial for determination of zearalenone (ZON) in baby food and animal feed was conducted. The study involved 39 participants in 16 European Union member states, as well as Turkey, Uruguay, and China, representing a cross-section of industry, and official food control and research institutes. The method is based on immunoaffinity column cleanup followed by high-performance liquid chromatography using fluorimetry (HPLC-FI). The test portion of the sample is extracted with methanol-water (75 + 25, v/v). The sample extract is filtered, diluted, and passed over an immunoaffinity column. ZON is eluted with methanol. The separation and determination of ZON is performed by reversed-phase HPLC-FI with an excitation wavelength of 274 nm and an emission wavelength of 446 nm. Test portions of the samples were spiked at levels of 20 and 30 microg/kg ZON in baby food and at levels of 100 and 150 microg/kg ZON in animal feed. Mean recoveries from each participant ranged from 78 to 119% with an average value of 92% for baby food and from 51 to 122% with an average value of 74% for animal feed. Based on results for spiked samples (blind duplicates at 2 levels), as well as naturally contaminated samples (blind duplicates at 3 levels), the relative standard deviation for repeatability (RSDr) in baby food ranged from 2.8 to 9.0%. For animal feed, this value ranged from 5.7 to 9.5%. The relative standard deviation for reproducibility (RSDR) in baby food ranged from 8.2 to 13.3%, and for animal feed this value ranged from 15.5 to 21.4%. The Horwitz ratio (HorRat) in baby food ranged from 0.3 to 0.4, and for animal feed this value ranged from 0.6 to 0.9. The method showed acceptable within- and between-laboratory precision for each matrix, as required by European legislation.     

A sensitive confirmatory method for aflatoxins in maize based on liquid chromatography/electrospray ionization tandem mass spectrometry

A liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for measurement of aflatoxins B1, B2, G1, and G2 in maize is described. Aflatoxins (AFs) were extracted from 1 g samples by using tri-portions of acetonitrile/water (80:20, v/v) (10 + 7 + 7 mL), and 2/5 of the extract diluted to 500 mL by water was cleaned up with a 100 mg Carbograph-4 cartridge. After the addition of the internal standard AFM1, the final extract was analyzed by LC/ESI-MS/MS in positive ion mode using multiple reaction monitoring with a triple-quadrupole instrument. A C(18) column thermostatted at 45 degrees C with a mobile phase gradient of acetonitrile/water with 2 mmol/L ammonium formate was used. Although the matrix suppression effect was negligible, quantitation was achieved by an external calibration procedure using matrix-matched standard solutions to improve accuracy. Sample recoveries at four spiking levels ranged from 81 to 101% (relative standard deviation (RSD) 相似文献   

Immunoaffinity column cleanup with liquid chromatography for determination of aflatoxin B1 in corn samples: interlaboratory study

An interlaboratory study was conducted to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatography (LC) method for the determination of aflatoxin B1 levels in corn samples, enforced by European Union legislation. A test portion was extracted with methanol-water (80 + 20); the extract was filtered, diluted with phosphate-buffered saline solution, filtered on a microfiber glass filter, and applied to an immunoaffinity column. The column was washed with deionized water to remove interfering compounds, and the purified aflatoxin B1 was eluted with methanol. Aflatoxin B1 was separated and determined by reversed-phase LC with fluorescence detection after either pre- or postcolumn derivatization. Precolumn derivatization was achieved by generating the trifluoroacetic acid derivative, used by 8 laboratories. The postcolumn derivatization was achieved either with pyridinium hydrobromide perbromide, used by 16 laboratories, or with an electrochemical cell by the addition of bromide to the mobile phase, used by 5 laboratories. The derivatization techniques used were not significantly different when compared by the Student's t-test; the method was statistically evaluated for all the laboratories. Five corn sample materials, both spiked and naturally contaminated, were sent to 29 laboratories (22 Italian and 7 European). Test portions were spiked with aflatoxin B1 at levels of 2.00 and 5.00 ng/g. The mean values for recovery were 82% for the low level and 84% for the high contamination level. Based on results for spiked samples (blind pairs at 2 levels) as well as naturally contaminated samples (blind pairs at 3 levels), the values for relative standard deviation for repeatability (RSDr) ranged from 9.9 to 28.7%. The values for relative standard deviation for reproducibility (RSDR) ranged from 18.6 to 36.8%. The method demonstrated acceptable within- and between-laboratory precision for this matrix, as evidenced by the HorRat values.     

Liquid chromatographic method for quantitation of patulin at 10 ng/mL in apple-based products intended for infants: interlaboratory study

An interlaboratory trial for the determination of patulin in apple juice and fruit puree was conducted, involving 17 participants representing a cross section of industry, official food control, and research facilities. Mean recoveries reported ranged from 74 (10 ng/g) to 62% (25 ng/g) for apple juice and from 72 (25 ng/g) to 74% (10 ng/g) for fruit puree. Based on results for spiked samples (blind pairs at 2 levels), as well as naturally contaminated samples (blind pairs at 3 levels), the relative standard deviation for repeatability (RSDr) in juice ranged from 8.0 to 14.3% and in puree from 3.5 to 9.3%. The relative standard deviation for reproducibility (RSD(R)) in juice ranged from 19.8 to 39.5% and in puree from 12.5 to 35.2%, reflecting HORRAT values from 0.6 to 1.0 for juice and 0.4 to 0.9 for puree. The method showed acceptable within-laboratory and between-laboratory precision for each matrix, as required by current European legislation.     

Determination of semicarbazide in fresh egg and whole egg powder by liquid chromatography/tandem mass spectrometry: interlaboratory validation study

An interlaboratory validation study was conducted according to harmonized protocols to evaluate the effectiveness of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method for the determination of semicarbazide (SEM) in fresh whole egg and in an industrially processed whole egg powder. The sample was extracted with hydrochloric acid and derivatized with 2-nitrobenzaldehyde, with 1,2-[(15)N2(13)C]SEM as the internal standard. The extract was neutralized and purified on a solid-phase extraction cartridge. SEM was determined by reversed-phase LC with detection by MS/MS. Five fresh egg samples, of which 3 were obtained from hens fed nitrofurazone (NFZ), one was spiked with SEM at 50 microg/kg and one was a blank sample, and 5 industrial whole egg powder samples, of which 3 were spiked with fresh whole egg from hens fed NFZ, one was spiked with SEM at 350 microg/kg, and one was a blank sample, were sent to 15 laboratories in 10 different European countries. Results were obtained from 12 participants. Average recoveries of SEM from the fresh egg and the egg powder samples were 105.3 and 121.3%, respectively. The relative standard deviation for repeatability (RSDr) ranged from 2.9 to 9.3%, and the relative standard deviation for reproducibility (RSDR) ranged from 22.5 to 38.1%. The method showed acceptable within- and between-laboratory precision for both matrixes, as evidenced by the HorRat values, at the target levels for the determination of SEM.     

Determination of eight sulfonamides in bovine kidney by liquid chromatography/tandem mass spectrometry with on-line extraction and sample clean-up

A sensitive, high performance liquid chromatography/tandem mass spectrometric (i.e. mass spectrometry/mass spectrometry; LC/MS/MS) method with on-line extraction and sample clean-up for the screening and confirmation of residues of sulfonamides in kidney is described. The sulfonamides are extracted from homogenized kidney with methanol. After centrifugation of the extract, an aliquot of the extract is directly injected on the LC/MS/MS system with further extraction and clean-up of the sample on-line. Detection of the analytes was achieved by positive electrospray ionization (ESI) followed by multiple reaction monitoring. For each sulfonamide the collisional decomposition of the protonated molecule to a common, abundant fragment ion was monitored. The method has been validated for sulfadimethoxine, sulfaquinoxaline, sulfamethazine, sulfamerazine, sulfathiazole, sulfamethoxazole, sulfadiazine and sulfapyridine. Calibration curves resulting from spiked blank kidney samples at the 10-200 microg/kg level showed good linear correlation. At the level of 50, 100 and 200 microg/kg both within- and between-day precision, as measured by relative standard deviation (RSD), were less than 16%. The limits of detection (LODs) ranged from 5 to 13.5 microg/kg. The recoveries ranged from 78 to 82%. The procedure provides a rapid, reliable and sensitive method for the determination of residues of sulfonamides in bovine kidney. The advantage of this method over existing methods is its decreased sample preparation and analysis time, which makes the method more suitable for routine analysis.     

Solvent effect on the determination of sulfamethazine by room-temperature photochemically induced fluorescence

Room-temperature photochemically-induced fluorescence (RTPF) was applied to the determination of sulfamethazine (SMT) in methanol, ethanol and 2-propanol. Optimal ultraviolet irradiation times ranged between 2 and 6 min. Linear calibration graphs were obtained over a concentration range of more than one order of magnitude. The relative standard deviations were within the range 1.4-2.2%. Limits of detection were between 40 and 80 ng/ml. The method was evaluated for its applicability to the analysis of SMT in pharmaceutical formulations.     

Determination of zearalenone in barley, maize and wheat flour, polenta, and maize-based baby food by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an affinity column cleanup liquid chromatography (LC) method for the determination of zearalenone (ZON) in a variety of cereals and cereal products at proposed European regulatory limits. The test portion is extracted with acetonitrile:water. The sample extract is filtered, diluted, and applied to an affinity column. The column is washed, and ZON is eluted with acetonitrile. ZON is quantified by reversed-phase LC with fluorescence detection. Barley, wheat and maize flours, polenta, and a maize-based baby food naturally contaminated, spiked, and blank (very low level) were sent to 28 collaborators in 9 European countries and 1 collaborator in New Zealand. Participants were asked to spike test portions of all samples at a ZON concentration equivalent to 100 microg/kg. Average recoveries ranged from 91-111%. Based on results for 4 artificially contaminated samples (blind duplicates) and 1 naturally contaminated sample (blind duplicate), the relative standard deviation for repeatability (RSDr) ranged from 6.9-35.8%, and the relative standard deviation for reproducibility (RSDR) ranged from 16.4-38.2%. The method showed acceptable within- and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values <1.7.