Determination of total soy isoflavones in dietary supplements, supplement ingredients, and soy foods by high-performance liquid chromatography with ultraviolet detection: collaborative study

An interlaboratory study was conducted to evaluate a method for determining total soy isoflavones in dietary supplements, dietary supplement ingredients, and soy foods. Isoflavones were extracted using aqueous acetonitrile containing a small amount of dimethylsulfoxide (DMSO) and all 12 of the naturally occuring isoflavones in soy were determined by high-performance liquid chromatography (HPLC) with UV detection using apigenin as an internal standard. Fifteen samples (6 pairs of blind duplicates plus 3 additional samples) of soy isoflavone ingredients, soy isoflavone dietary supplements, soy flour, and soy protein products were successfully analyzed by 13 collaborating laboratories in 6 countries. For repeatability, the relative standard deviations (RSDr) ranged from 1.07 for samples containing over 400 mglg total isoflavones to 3.31 for samples containing 0.87 mg/g total isoflavones, and for reproducibility the RSDR values ranged from 2.29 for samples containing over 400 mg/g total isoflavones to 9.36 for samples containing 0.87 mg/g total isoflavones. HorRat values ranged from 1.00 to 1.62 for all samples containing at least 0.8 mg/g total isoflavones. One sample, containing very low total isoflavones (< 0.05 mg/g), gave RSDR values of 175 and a HorRat value of 17.6. This sample was deemed to be below the usable range of the method. The method provides accurate and precise results for analysis of soy isoflavones in dietary supplements and soy foods.     

Determination of hydrastine and berberine in goldenseal raw materials, extracts, and dietary supplements by high-performance liquid chromatography with UV: collaborative study

A multilaboratory collaborative study was conducted on a high-performance liquid chromatographic (HPLC) method utilizing UV detection, previously validated using AOAC single-laboratory validation guidelines for determination of hydrastine and berberine in goldenseal (Hydrastis canadensis L.) raw materials, extracts, and dietary supplements at levels ranging from 0.4 to 6% (w/w). Nine collaborating laboratories determined the hydrastine and berberine content in 8 blind samples. Sample materials included powdered botanical raw materials, whole root material, and 4 finished product dietary supplements containing either goldenseal powdered root material or extract. The materials were extracted with an acidified water and acetonitrile solution. HPLC analyses of the extracts were performed on a C18 column using UV detection at 230 nm. Results for powdered root material and capsule products ranged from about 0.2% (w/w) for each alkaloid to about 4% (w/w) for each alkaloid. Liquid tincture results were approximately 4000-5000 microg/mL for each alkaloid. Reproducibility relative standard deviations (RSDR) for hydrastine ranged from 2.68 to 6.65%, with HorRat values ranging from 0.77 to 1.89. RSDR for berberine ranged from 5.66 to 7.68%, with HorRat values ranging from 1.32 to 2.12. All finished products containing goldenseal extract yielded HorRat values <2.0. Based on these results, the method is recommended for Official First Action for determination of hydrastine and berberine in goldenseal raw materials and dietary supplement finished products containing powdered goldenseal and goldenseal extract.     

Determination of monensin, narasin, and salinomycin in mineral premixes, supplements, and animal feeds by liquid chromatography and post-column derivatization: collaborative study

A liquid chromatographic (LC) method for the analysis of monensin, narasin, and salinomycin in mineral premixes, supplements, and complete animal feeds at medicating and trace levels was collaboratively studied. The method uses methanol-water (90 + 10) extraction with mechanical shaking for 1 h, filtration, and dilution if necessary. Determination of the 3 ionophores is by reversed-phase LC using post-column derivatization with vanillin and detection at 520 nm. Suspect positive trace-level products and medicated feeds containing unexpected ionophores are confirmed by hexane extraction or post-column derivatization with dimethylaminobenzaldehyde (DMAB). Twenty-five test samples of medicated feeds, supplements, and mineral and drug premixes, and 9 test samples for trace-level analysis were sent to 11 collaborators in Bulgaria, Czech Republic, Portugal, France, The Netherlands, United States, and Canada. Acceptable results were received from 10 laboratories. For the medicated complete feeds, supplements, and mineral premixes, RSDr values (within-laboratory repeatability) ranged from 2.5 to 5.2%, RSDR values (among-laboratory reproducibility) ranged from 2.7 to 6.8%, and HorRat values ranged from 0.31 to 1.30. For the drug premixes, the result variability was excessive and HorRat values ranged from 2.27 to 14.1. For the trace-level test samples, all laboratories correctly identified the analytes and did not report any false positives. RSDr values ranged from 1.3 to 9.5%, RSDR values ranged from 5.2 to 13.1%, and HorRat values ranged from 0.4 to 0.97.     

Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, natural colorants, and wines by the pH differential method: collaborative study

This collaborative study was conducted to determine the total monomeric anthocyanin concentration by the pH differential method, which is a rapid and simple spectrophotometric method based on the anthocyanin structural transformation that occurs with a change in pH (colored at pH 1.0 and colorless at pH 4.5). Eleven collaborators representing commercial laboratories, academic institutions, and government laboratories participated. Seven Youden pair materials representing fruit juices, beverages, natural colorants, and wines were tested. The repeatability relative standard deviation (RSDr) varied from 1.06 to 4.16%. The reproducibility relative standard deviation (RSDR) ranged from 2.69 to 10.12%. The HorRat values were < or = 1.33 for all materials. The Study Director recommends that the method be adopted Official First Action.     

Determination of phytase activity in feed by a colorimetric enzymatic method: collaborative interlaboratory study.

Fourteen laboratories participated in a collaborative study (coded fyt9404) and 13 laboratories participated in a study (coded fyt9410) to validate a colorimetric assay for determination of microbial phytase activity in feed. For each study, all laboratories received 6 laboratory samples provided by one commercial supplier (phytase activity levels within the range of 200-400 per kg) to be analyzed in duplicate. Method performance was calculated and statistical calculations were executed according to AOAC guidelines. Results from 3 laboratories for study fyt9404 and from one laboratory for study fyt9410 were excluded from statistical analysis because of invalid data determined during initial review by Youden pair, value versus laboratory. For study fyt9404, repeatability relative standard deviation (RSDr) values ranged from 6.2 to 8.6%, and reproducibility relative standard deviation (RSDR) values ranged from 14.1 to 27.6%. No outliers were identified. For study fyt9410, RSDr values ranged from 3.9 to 7.9%, and RSDR values ranged from 14.0 to 20.5%. With outliers excluded, RSDr values ranged from 2.5 to 7.9%, and RSDR values ranged from 14.0 to 20.5%.     

Measurement of resistant starch by enzymatic digestion in starch and selected plant materials: collaborative study

Interlaboratory performance statistics was determined for a method developed to measure the resistant starch (RS) content of selected plant food products and a range of commercial starch samples. Food materials examined contained RS (cooked kidney beans, green banana, and corn flakes) and commercial starches, most of which naturally contain, or were processed to yield, elevated RS levels. The method evaluated was optimized to yield RS values in agreement with those reported for in vivo studies. Thirty-seven laboratories tested 8 pairs of blind duplicate starch or plant material samples with RS values between 0.6 (regular maize starch) and 64% (fresh weight basis). For matrixes excluding regular maize starch, repeatability relative standard deviation (RSDr) values ranged from 1.97 to 4.2%, and reproducibility relative standard deviation (RSDR) values ranged from 4.58 to 10.9%. The range of applicability of the test is 2-64% RS. The method is not suitable for products with <1% RS (e.g., regular maize starch; 0.6% RS). For such products, RSDr and RSDR values are unacceptably high.     

Evaluation of a method to determine flavonol aglycones in Ginkgo biloba dietary supplement crude materials and finished products by high-performance liquid chromatography: collaborative study

An interlaboratory study was conducted for evaluation of a method to determine the flavonol aglycones quercetin, kaempferol, and isorhamnetin in Ginkgo biloba products. The method calculates total glycosides based on these aglycones formed after acid hydrolysis. Twelve matrixes were chosen for study by 12 collaborating laboratories in 2 countries. Test materials included crude leaf material, standardized dry powder extract, single and multiple entity finished products, ethanol and glycerol tinctures, and National Institute of Standards and Technology (NIST) standard reference materials (SRMs). Results from 11 laboratories were used for the final calculations. Eight of the 12 matrixes evaluated produced acceptable results for total flavonol glycosides, with HorRat scores ranging from 1.31 to 2.05; repeatability relative standard deviations (RSDr) from 1.46 to 4.14; and reproducibility relative standard deviations (RSDR) from 4.67 to 9.69. These 8 matrixes consisted primarily of simple dosage forms (e.g., dry powder extracts, crude leaf samples, liquid extracts, and SRMs) and a single tablet product (Ginkgo Awareness). Four additional matrixes, consisting of 3 tablets and 1 soft gel product (Ginkgold, Ginkoba, Ginkogen, and Ginkgo Phytosome, respectively), showed greater total flavonol glycoside HorRat scores in comparison, ranging from 2.39 to 5.13, with RSDr values from 2.83 to 8.16, and RSDR values from 8.53 to 20.4. Based on the results presented here, the method is recommended for Official First Action for determination of total flavonol glycosides calculated from quercetin, kaempferol, and isorhamnetin in dry powder extracts, crude leaf material, liquid extracts, and a select finished product, Ginkgo Awareness.     

Determination of lasalocid sodium in animal feeds and premixes by reversed-phase liquid chromatography: collaborative study

A liquid chromatographic (LC) method for the analysis of lasalocid sodium in premixes, complete animal feeds, and trace-level feeds was collaboratively studied. The method employs a 0.5% HCI acidified methanol extraction followed by 20 min sonication in a water bath heated to 40 degrees C. Samples are then shaken on a mechanical shaker for 1 h and stored overnight, followed by an additional 10 min shaking the following morning. Sample extracts are diluted if necessary with extractant, filtered, and injected onto an LC system. Determination of all lasalocid homologs is by reversed-phase LC with fluorescence detection at 314 nm excitation and 418 nm emission. Eight samples of drug premixes, medicated feeds, and mineral supplements, along with 2 samples for trace-level analysis were sent to 20 collaborators in the United States, Canada, and The Netherlands. Study data were returned by 17 laboratories. Two additional supplemental trace-level samples and a blank feed were provided to 15 of the collaborating laboratories, and test data were received from all 15 participants. For the drug premixes, medicated feeds, and mineral supplements, RSDr values (within-laboratory repeatability) ranged from 1.2 to 19.9%, RSDR values (among-laboratory reproducibility) ranged from 3.4 to 32.3%, and HorRat values ranged from 0.35 to 3.73. For the trace-level samples, only lasalocid A, the predominant homolog comprising > 90% of the sum of all homolog peak area, was quantified. All laboratories correctly identified the analyte. Although some instrument response was reported by a number of laboratories for the blank feed, all but one laboratory's results were well below the 1 mg/kg limit of quantification. RSDr values for the initial 2 trace-level samples were excessive, ranging from 51.6 to 64.4%. RSDR values ranged from 51.6 to 75.7%, and HorRat values ranged from 3.6 to 4.0. Data for the initial trace-level samples indicated that the test samples were improperly prepared to ensure homogeneity, and a new set of supplemental samples was provided to collaborators, with significantly improved results. RSDr values for the 2 supplemental trace-level samples ranged from 1.6 to 2.5%, RSDR values ranged from 5.6 to 9.2%, and HorRat values ranged from 0.43 to 0.62.     

Determination of coenzyme Q10 content in raw materials and dietary supplements by high-performance liquid chromatography-UV: collaborative study

An international collaborative study was conducted of a high-performance liquid chromatographic (HPLC)-UV method for the determination of coenzyme Q10 (CoQ10, ubidecarenone) in raw materials and dietary supplements. Ten collaborating laboratories determined the total CoQ10 content in 8 blind duplicate samples. Sample materials included CoQ10 raw material and 4 finished product dietary supplements representing softgels, hardshell gelatin capsules, and chewable wafers. In addition, collaborating laboratories received a negative control and negative control spiked with CoQ10 at low and high levels to determine recovery. Materials were extracted with an acetonitrile-tetrahydrofuran-water mixture. Ferric chloride was added to the test solutions to ensure all CoQ10 was in the oxidized form. The HPLC analyses were performed on a C18 column using UV detection at 275 nm. Repeatability relative standard deviations (RSDr) ranged from 0.94 to 5.05%. Reproducibility relative standard deviations (RSDR) ranged from 3.08 to 17.1%, with HorRat values ranging from 1.26 to 5.17. Recoveries ranged from 74.0 to 115%. Based on these results, the method is recommended for Official First Action for determination of CoQ10 in raw materials and dietary supplement finished products containing CoQ10 at a concentration of >100 mg CoQ10/g test material.     

Determination of aristolochic acid I in botanicals and dietary supplements potentially contaminated with aristolochic acid I using LC-UV with confirmation by LC/MS: collaborative study

An interlaboratory study was conducted to evaluate a method for the determination of aristolochic acid I, also known as aristolochic acid A, at levels > 2.00 microg/g in botanical species and dietary supplements potentially contaminated with aristolochic acid I. Aristolochic acid I was extracted from various matrixes with aqueous acetonitrile. The amount of aristolochic acid I present was determined by liquid chromatography (LC) using an ultraviolet (UV) detector with confirmation by LC/mass spectrometry (MS). Thirteen blind duplicates were successfully analyzed by 10 collaborators, and aristolochic acid I was successfully confirmed in 1 blind duplicate by 8 collaborators. For repeatability, the relative standard deviation (RSD(r)) ranged from 1.72 to 16.3% and for reproducibility, the RSDR ranged from 5.42 to 19.8%. HorRat values were not applicable for 2 materials but varied from 0.7 to 1.8 for 11 materials. Each collaborating laboratory had calibration curves with correlation coefficients > 0.998. In addition, all of the collaborators that conducted the confirmation were able to verify the identity of aristolochic acid I using LC/MS/MS (using either ion trap or triple quad).     

Determination of sodium in foods by flame atomic absorption spectrometry after microwave digestion: NMKL interlaboratory study

Nine laboratories participated in an interlaboratory method performance (collaborative) study of a method for the determination of sodium in foods by flame atomic absorption spectrometry after wet digestion, using a microwave oven technique. Before the study, the laboratories were able to practice on samples with defined sodium levels (pretrial test). The method was tested on a total of 6 foods (broccoli, carrot, bread, saithe fillet, pork, and cheese) with sodium concentrations of 1480-8260 mg/kg. The materials were presented to the participants in the study as blind duplicates, and the participants were asked to perform single determinations for each sample. The repeatability relative standard deviations (RSDr) for sodium ranged from 1.9 to 6.5%. The reproducibility relative standard deviations (RSDR) ranged from 4.2 to 6.9%. The HorRat values ranged from 0.9 to 1.6.     

Determination of moisture and fat in meats by microwave and nuclear magnetic resonance analysis: collaborative study

Ten laboratories participated in a collaborative study to determine the total moisture and fat in raw and processed meat products by microwave drying and nuclear magnetic resonance (NMR) spectroscopy. Meat products were prepared following the AOAC Method and analyzed using CEM Corp.'s SMART Trac Moisture and Fat Analysis system. SMART Trac provides moisture results by measuring the weight loss on drying by microwave energy. The dried sample is then analyzed by NMR spectrometry for fat content. Moisture and fat results are displayed and reported by the SMART Trac as a percentage (g/100 g). Microwave drying is an AOAC-approved reference method (Method 985.14), Moisture in Meat and Poultry Products. NMR spectrometry is a secondary technique used to determine the concentration of various constituents in biological, organic, or chemical samples. The study design was based on Youden's matched pair principle for collaborative tests. For the purposes of this study, 10 laboratories each tested 10 Youden matched pairs, for a total of 20 samples. The study samples represented a range of products processed daily in plant operations. Included were raw meat samples (beef, pork, chicken, and turkey) as well as processed meats (beef hot dog, pork sausage, and ham). The total moisture content of the undiluted samples, as received for the purposes of this study, was determined by AOAC Method 950.46 and ranged from 54.03 to 74.99%. The total fat content of the undiluted samples was determined by AOAC Method 960.39 and ranged from 1.00 to 29.79%. Statistical analysis of study results for total moisture yielded a relative standard deviation for repeatability (RSDr) range of 0.14 to 0.95% and a relative standard deviation for reproducibility (RSDR) range of 0.26 to 0.95%. Statistical analysis for total fat yielded similar RSDr and RSDR range of 0.74 to 4.08%. Results for turkey had higher RSDr and RSDR values, both at 12.6%, due to low fat content and possibly to the separation of the samples observed by some of the collaborators. Results demonstrate that microwave drying with NMR is a rapid, practical method providing results equivalent to AOAC Methods 950.46 (Forced Air Oven Drying) and 960.39 (Soxhlet Ether Extraction) in raw and processed meat products.     

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.     

Method for the rapid determination of protein in meats using the CEM Sprint protein analyzer: collaborative study

A collaborative study was conducted to determine the protein content of raw and processed meat products by a protein-tagging and colorimetric technique. Meat products were prepared following AOAC Official Method 983.18 and analyzed using CEM Corporation's Sprint Rapid Protein Analyzer. Sprint provides protein results by combining an accurately weighed test portion with a known amount of dye-binding agent. The dye-binding agent binds with the lysine, histidine, and arginine, as well as the n-terminus of the proteins commonly found in raw meat and processed meat products. Results are displayed and reported by the Sprint as a percentage (g/100 g) of protein. Ten blind duplicate study samples were sent to 10 collaborating laboratories in the United States. The within-laboratory (repeatability) relative standard deviation (RSD(r)) ranged from 0.91 to 3.04%, and between-laboratories (reproducibility) relative standard deviation (RSDR) ranged from 1.50 to 3.41% for protein. The method is recommended for Official First Action.     

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 neutral lactase activity in industrial enzyme preparations by a colorimetric enzymatic method: collaborative study

Thirteen laboratories participated in a collaborative study to validate a colorimetric assay for determining neutral lactase activity in industrial enzyme preparations. Each laboratory received 5 duplicate samples with activity levels of 2000 and 5000 neutral lactase units provided by 4 commercial suppliers. Two laboratories did not return results. Method performance was calculated according to AOAC guidelines. From the 11 remaining laboratories, 3 were excluded from statistical analysis because of invalid data determined during initial review by Youden pair, value versus laboratory. Repeatability relative standard deviation (RSDr) values ranged from 3.20 to 8.62%, and reproducibility relative standard deviation (RSDR) values ranged from 8.77 to 16.35%. With outliers excluded, RSDr values ranged from 2.94 to 5.01%, and RSDR values ranged from 7.50 to 13.84%. The colorimetric enzymatic method for determining neutral lactase activity in industrial enzyme preparations has been adopted first action by AOAC INTERNATIONAL.     

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.     

Determination of ginsenosides (ginseng saponins) in dry root powder from Panax ginseng, Panax quinquefolius, and selected commercial products by liquid chromatography: interlaboratory study

Twelve collaborating laboratories assayed 4 products, namely, Panax ginseng, Panax quinquefolius, and 2 ginseng products, for 6 ginsenosides: Rb1, Rb2, Rc, Rd, Re, and Rg1. Collaborators also received a negative control for the recovery study. Pure ginsenosides were provided as reference standards for the liquid chromatography (LC) analysis and the system suitability tests. The LC analyses were performed on the methanol extract using UV detection at 203 nm. For P. ginseng, individual ginsenosides were consistent in their means; repeatability standard deviations (RSDr) ranged from 4.17 to 5.09% and reproducibility standard deviations (RSDR) ranged from 7.27 to 11.3%. For P. quinquefolius, the Rb1 and Rb2 ginsenosides were higher and lower in concentration than P. ginseng, with RSDr values of 3.44 and 6.60% and RSDR values of 5.91 and 12.6% respectively, and other analytes at intermediate precisions. For ginseng commercial products, RSDr values ranged from 3.39 to 8.12%, and RSDR values ranged from 7.65 to 16.5%. A recovery study was also conducted for 3 ginsenosides: Rg1, Re, and Rb1. The average recoveries were 99.9, 96.2, and 92.3%, respectively. The method is not applicable for the determination of Rg1 and Re in ginseng product at levels <300 mg/kg.     

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.