Determination of Tilmicosin in Bovine, Swine, Chicken, and Turkey Tissues by Liquid Chromatography With Tandem Mass Spectrometry, Single-Laboratory Validation.

BACKGROUND: An LC-MS/MS method was developed for determination and confirmation of tilmicosin in bovine, swine, chicken, and turkey tissues (liver, kidney, muscle, and skin/fat) and bovine milk. OBJECTIVE: The method was subjected to single-laboratory validation to establish method performance parameters. METHOD: Animal tissues and bovine milk were fortified at four concentrations ranging from 0.5 times the lowest maximum residue limit (MRL) or tolerance to 2 times the highest MRL or tolerance considering the Codex and EU MRLs and the US tolerances in the various tissues and milk studied. Incurred tissues were analyzed to verify the precision of the method. RESULTS: The data demonstrated linearity of matrix-matched calibration curves using a weighted (1/×) regression. Recoveries varied from 83.3 to 107.1%. Repeatability precision (RSDr) ranged from 0.465 to 13.4% and intermediate precision (RSDi) ranged from 2.24 to 14.7% in fortified tissue. Repeatability of the method was verified in incurred tissues, ranging from 3.41 to 16.0%. The limits of detection and quantitation of the method are presented and vary by matrix. One confirmatory transition ion was examined across all matrixes and met US and EU criteria for mass spectrometry confirmation. The method was shown to be robust when small changes in method parameters were made, and stability of the analyte in fortified tissues, extracts, standard solutions, and matrix-matched standards was estimated. CONCLUSIONS: The data satisfy the requirements of the AOAC Stakeholder Panel for Veterinary Drug Residue Methods for single-laboratory validation studies and the U.S. Food and Drug Administration Center for Veterinary Medicine Guidance for Industry #208 (VICH GL49). HIGHLIGHTS: The LC-MS/MS method was demonstrated to be suitable for determination and confirmation of tilmicosin residues in bovine, swine, chicken, and turkey tissues and bovine milk based on Codex and EU MRLs and US tolerances.

Determination of Lactose in Lactose-Free and Low-Lactose Milk, Milk Products, and Products Containing Dairy Ingredients by the LactoSens®R Amperometry Method: Final Action 2020.01.

BACKGROUND: The LactoSens®R method was previously shown to have acceptable accuracy and repeatability precision as required by AOAC Standard Method Performance Requirements (SMPR®) 2018.009 for determination of lactose in low-lactose or lactose-free milk, milk products, and products containing dairy ingredients and was awarded Official Method of AnalysisSM (OMA) First Action status in 2020. OBJECTIVE: The method was subjected to a multilaboratory validation (MLV) study to evaluate the reproducibility precision of the method. METHODS: Fourteen validation materials were provided to 15 laboratories in seven countries as blind duplicates. The materials ranged from 0 to 173 mg/100 g lactose. Each laboratory analyzed the blind duplicates according to OMA 2020.01. The data were analyzed for repeatability and reproducibility precision. RESULTS: RSDr values varied from 2.81 to 8.76%, and RSDR values varied from 4.25 to 12.5%. When sorted by category and concentration range, these results met the repeatability and reproducibility criteria required by SMPR 2018.009. CONCLUSIONS: The data generated in the MLV support the adoption of OMA 2020.01 as Final Action status. HIGHLIGHTS: The LactoSensR method, as described by OMA 2020.01, provides an accurate and precise determination of lactose in a variety of low-lactose and lactose-free milk, milk products, and products containing dairy ingredients in minutes.

Determination and Identification of Nicarbazin, Measured as 4,4'-Dinitrocarbanilide (DNC), in Chicken Tissues by Liquid Chromatography With Tandem Mass Spectrometry: Final Action 2013.07.

BACKGROUND: AOAC Method 2013.07 was adopted as First Action in 2013. Since then, the method has been used in numerous residue depletion studies with favorable comments from analysts. OBJECTIVE: To analyze data from residue depletion studies to support Final Action status. METHOD: Ten residue depletion studies were conducted during May 2014 through May 2019. For each study, harvested incurred tissues were analyzed for nicarbazin using AOAC Method 2013.07 in 1 of 4 laboratories. Each analytical run included one or more fortified quality control test portions. The data from these known fortified matrix test portions were analyzed for reproducibility and repeatability. RESULTS: For muscle tissues, relative recovery was 90.4% (95% CI 83.8 to 97.5); RSDr was 5.4% (95% CI 3.8 to 9.2); and RSDR was 7.9%. In the liver, values were 94.5% (95% CI 91.1 to 98.0), 5.8% (95% CI 4.1 to 9.9), and 6.8%, respectively. In the kidney, values were 91.5% (95% CI 85.3 to 98.1), 5.2% (95% CI 3.7 to 8.8), and 9.0%, respectively. In skin with adhering fat, values were 94.5% (95% CI 89.2 to 100.1), 8.9% (95% CI 6.3 to 15.1), and 8.9%, respectively. In all cases, repeatability and reproducibility were within acceptable limits. CONCLUSIONS: The data and positive feedback support the transition of AOAC Method 2013.07 from First Action to Final Action. HIGHLIGHTS: Final action status is supported by data collected during routine use of the method rather than a traditional multi-laboratory collaborative study. Data were subjected to statistical analysis using the pC-metamer, and then transformed back to the traditional C-metamer.

Comparison of the Modified Centers for Disease Control and Prevention 2019-Novel Coronavirus Real-Time RT-PCR Method for Detection of Infectious and Heat-Inactivated Virus on Stainless Steel.

BACKGROUND: Infectious Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) was used in the validation of methods for detection of SARS-CoV-2 on stainless-steel surfaces in the AOAC Research Institute Emergency Response Validation project. Handling infectious virus requires Biosafety Level (BSL)-3 facilities. OBJECTIVE: To compare the recovery and detection of infectious and heat-inactivated (HI; 65°C for 30 min) SARS-CoV-2 from stainless steel by the modified US Centers for Disease Control and Prevention (CDC) 2019-Novel Coronavirus (2019-nCoV) Real Time Reverse Transcription Polymerase Chain Reaction (RT-PCR) Diagnostic Panel. METHOD: Viral stocks were diluted in viral transport medium (VTM) and deposited onto stainless-steel test areas at 2 × 103 and 2 × 104 genomic copies for low and high, respectively. Test areas were sampled and aliquots of the resulting test solutions analyzed by RT-qPCR according to the CDC method. Results were analyzed by probability of detection (POD) statistics. RESULTS: The low level, where fractional positive results (25-75%) are expected, yielded PODI = 0.80 (0.58, 0.92) for the infectious virus and PODHI = 0.15 (0.05, 0.36) for the HI virus. The bias, dPODHI = -0.65 (-0.80, -0.35), demonstrated a statistical difference between infectious and HI virus detection. No difference was observed at the high inoculation level. CONCLUSIONS: Despite the statistical difference observed, the use of the HI virus is a viable alternative for matrix extension studies using a method comparison study design. Highlights: The use of HI SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies. HIGHLIGHTS: The use of HI SARS-CoV-2 can mitigate the need for a BSL-3 facility for matrix extension validation of alternative methods in SARS-CoV-2 studies.

Determination of Minerals and Trace Elements in Milk, Milk Products, Infant Formula, and Adult Nutrition: Collaborative Study 2011.14 Method Modification.

Official MethodSM 2011.14/ISO 15151:2018/IDF 229:2018 uses microwave digestion of samples and inductively coupled plasma-atomic emission spectrometry for determination of nine elements, including Ca, Cu, Fe, K, Mg, Mn, Na, P, and Zn. The method was evaluated in a collaborative study of 25 products, including 13 fortified nutritional products (powders, ready-to-feed liquids, and liquid concentrates), five product placebos, six dairy products (liquids, powders, butter, and processed cheese), and the National Institute for Standards and Technology (NIST) Standard Reference Material (SRM) 1849a, in compliance with AOAC INTERNATIONAL Standard Method Performance Requirement (SMPR®) 2014.004. This study significantly expanded the applicability of Official Method 2011.14 beyond the original scope of chocolate milk powder, dietetic milk powder, infant cereal, peanut butter, and wheat gluten. The study included 14 collaborators from 11 countries, and results were compared to SMPR 2014.004. Accuracy of the method was demonstrated using NIST SRM 1849a, yielding recoveries across all laboratories of 98-101% for the nine elements. Precision for the 13 fortified nutritional product samples was 2.2-3.9% for repeatability (relative SD of repeatability) and 6.0-12.2% for reproducibility (RSDR). Excluding Mn, which was present at a wide range of concentrations, the reproducibility was 6.0-9.5%, meeting the performance requirements of SMPR 2014.004. Placebo samples (not fortified with Cu, Fe, Mn, or Zn) yielded acceptable repeatability of 1.8-2.9% for Ca, K, Mg, Na, and P (minerals) but 5.4-29.4% for the low levels of Cu, Fe, Mn, and Zn (trace elements). Reproducibility for the placebos showed the same pattern, with acceptable reproducibility (5.4-10.3%) for minerals but not for the low levels of the trace elements (13.2-82.8%). In the six dairy product samples, repeatability ranged from 1.6 to 3.6% for the minerals, Zn, and the low range of Mn but from 9.4 to 24.6% for Cu, Fe, and the high range of Mn, where concentrations were low as for the nutritional placebos. Reproducibility in the dairy samples was 5.3-8.8% for the minerals but 11.4-55.0% for the trace elements. The mean concentrations of Cu, Fe, and Zn in the dairy products were similar with those in the placebo products, while Zn was present at levels more similar with the fortified nutritional products. Thus, the method met the SMPR criteria except where the trace minerals were present at very low levels. Based on these results, the AOAC Stakeholder Panel for Infant Formula and Adult Nutritionals recommended Final Action status of the expanded applicability of the method. The method was adopted as Final Action by the AOAC Official Methods Board.

Determination of Total Phenolic Content Using the Folin-C Assay: Single-Laboratory Validation, First Action 2017.13.

A single-laboratory validation of a method using Folin & Ciocalteu's phenol reagent (Folin-C reagent) for determination of total phenolic content of selected dietary supplement extracts was performed. The method is composed of a water extraction of dried extracts with sonication followed by reaction with the Folin-C reagent. The resulting colorimetric reaction is measured at 765 nm and compared with a standard curve generated with gallic acid standard solutions. The validation results were compared with Standard Method Performance Requirement (SMPR®) 2015.009, developed by the Stakeholder Panel on Dietary Supplements. The method demonstrated acceptable within-day RSDr of 1.96-7.47% for the five matrixes studied (grape seed extract, grape skin extract, black tea extract, green coffee extract, and cocoa extract). When gallic acid was spiked into maltodextrin (a surrogate dietary supplement carrier) at 30 or 70%, the recovery ranged from 91 to 104%, within the acceptable range established by SMPR 2015.009. Selectivity testing with glucose, fructose, and sucrose demonstrated no positive interference by these compounds. Finally, ruggedness studies demonstrated no significant effects due to changes in the heating apparatus, test material weight, read time after reaction, amount of Folin-C reagent, reaction time, reaction temperature, and amount of Na2CO3. The single-laboratory validation results support adoption of the method as First Action Official MethodSM 2017.13 and further evaluation in a collaborative study.

Determination of Monensin in Bovine Tissues: A Bridging Study Comparing the Bioautographic Method (FSIS CLG-MON) with a Liquid Chromatography-Tandem Mass Spectrometry Method (OMA 2011.24).

The U.S. Department of Agriculture, Food Safety Inspection Service regulatory method for monensin, Chemistry Laboratory Guidebook CLG-MON, is a semiquantitative bioautographic method adopted in 1991. Official Method of AnalysisSM (OMA) 2011.24, a modern quantitative and confirmatory LC-tandem MS method, uses no chlorinated solvents and has several advantages, including ease of use, ready availability of reagents and materials, shorter run-time, and higher throughput than CLG-MON. Therefore, a bridging study was conducted to support the replacement of method CLG-MON with OMA 2011.24 for regulatory use. Using fortified bovine tissue samples, CLG-MON yielded accuracies of 80-120% in 44 of the 56 samples tested (one sample had no result, six samples had accuracies of >120%, and five samples had accuracies of 40-160%), but the semiquantitative nature of CLG-MON prevented assessment of precision, whereas OMA 2011.24 had accuracies of 88-110% and RSDr of 0.00-15.6%. Incurred residue results corroborated these results, demonstrating improved accuracy (83.3-114%) and good precision (RSDr of 2.6-20.5%) for OMA 2011.24 compared with CLG-MON (accuracy generally within 80-150%, with exceptions). Furthermore, χ2 analysis revealed no statistically significant difference between the two methods. Thus, the microbiological activity of monensin correlated with the determination of monensin A in bovine tissues, and OMA 2011.24 provided improved accuracy and precision over CLG-MON.

Determination of Withanolides in Withania somnifera by Liquid Chromatography: Single-Laboratory Validation, First Action 2015.17.

An LC method was developed and validated in 2007 for analyzing Withania somnifera raw material (root) and dried extracts for withanolide content, including withanoside IV, withanoside V, withaferin A, 12-deoxywithastromonolide, withanolide A, and withanolide B. The method involved the extraction of the analytes with methanol, their subsequent filtration, and then analysis on a C18 column with an acetonitrile gradient and UV detection. Single-laboratory validation yielded linearity generally in the range of 20 to 200 µg/mL for each analyte, with a repeatability precision of RSD < 3% in most cases, and recovery in the range of 90 to 105%. These results compare well with the performance criteria recently detailed in AOAC Standard Method Performance Requirement 2015.007. The method was shown to be rugged with respect to different analysts, equipment, and days of analysis, and the sample solution was shown to be stable for 24 h at room temperature after extraction. The method was reviewed by the AOAC Expert Review Panel on Dietary Supplements (Set 2 Ingredients) and approved for First Action Official MethodSM status.

Determination of Narasin in Chicken Fat: A Bridging Study Comparing the Bioautographic Method (FSIS CLG Method R22) to a Liquid Chromatography with Tandem Mass Spectrometry Method (AOAC Method 2011.24).

Lilly Method AM-AA-CA-R108-AB-755, which is substantially the same as U.S. Department of Agriculture, Food Safety and Inspection Service (FSIS) Chemistry Laboratory Guidebook (CLG) method R22, is the current regulatory method for determining narasin in cattle and chicken tissues and is based on bioautography, creating a zone of inhibition of bacterial growth, with the size of the zone correlating to the amount of narasin extracted from the tissue. AOAC Method 2011.24 is an LC-tandem mass spectrometry (MS/MS) method for determining narasin content from bovine, swine, or chicken tissues. It has many advantages over the regulatory method, including higher throughput, less solvent use, no use of carbon tetrachloride, a wider method range, inclusion of swine tissues, and it is less labor intensive. In this study, AOAC Method 2011.24 was compared to FSIS CLG method R22 for the determination of narasin in chicken abdominal fat. Fortified chicken-fat samples ranging from 20 to 960 ng/g and incurred chicken-fat samples ranging from 40 to 480 ng/g were assayed by both methods in triplicate. Mean accuracies for the two methods were similar, 77-110% for CLG R22 and 84-96% for AOAC Method 2011.24, and the method results showed a linear correlation. The methods differed in precision, however, with the CLG R22 method yielding 2.6-34% RSD and AOAC Method 2011.24 yielding 0.15-6.4% RSD. It is recommended that AOAC Method 2011.24-granted AOAC Official Method(SM) Final Action status-be adopted as the official U.S. regulatory method.

Determination of Chloride in Infant Formula and Adult/Pediatric Nutritional Formula by Potentiometric Titration: Single-Laboratory Validation, First Action 2015.07.

A potentiometric method for determination of chloride was validated against AOAC Standard Method Performance Requirement (SMPR(®)) 2014.015. Ten AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) matrixes, including National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1849a, were tested in duplicate on 6 independent days. The repeatability (RSDr) ranged from 0.43 to 1.34%, and the intermediate reproducibility (RSDiR) ranged from 0.80 to 3.04%. All results for NIST SRM 1849a were within the range of the certified concentration (701 ± 17 mg/100 g). Recovery was demonstrated with two overspike levels, 50 and 100%, in the 10 SPIFAN matrixes. Samples were tested in duplicate on 3 different days, and all results were within the SMPR requirement of 95 to 105%. The LOQs of the method for powdered products and ready-to-feed or reconstituted products were 20 mg/100 g and 2.2 mg/100 mL, respectively. A wide analytical range from the LOQ to 99.5% chlorine content can be reached with an appropriate dilution factor, but in practice, the upper analytical value observed in routine matrix testing was approximately 1080 mg/100 g in skim milk powder. This is a rapid, simple, and reliable chlorine-testing method applicable to infant formula, adult nutritionals, and ingredients used in these dairy-based products, such as skim milk powder, desalted whey powder, whey protein powder, and whole milk powder.

Determination of Chondroitin Sulfate Content in Raw Materials and Dietary Supplements by High-Performance Liquid Chromatography with UV Detection After Enzymatic Hydrolysis: Single-Laboratory Validation First Action 2015.11.

A previously validated method for determination of chondroitin sulfate in raw materials and dietary supplements was submitted to the AOAC Expert Review Panel (ERP) for Stakeholder Panel on Dietary Supplements Set 1 Ingredients (Anthocyanins, Chondroitin, and PDE5 Inhibitors) for consideration of First Action Official Methods(SM) status. The ERP evaluated the single-laboratory validation results against AOAC Standard Method Performance Requirements 2014.009. With recoveries of 100.8-101.6% in raw materials and 105.4-105.8% in finished products and precision of 0.25-1.8% RSDr within-day and 1.6-4.72% RSDr overall, the ERP adopted the method for First Action Official Methods status and provided recommendations for achieving Final Action status.

Determination of Minerals and Trace Elements in Infant Formula and Adult/Pediatric Nutritional Formula by Inductively Coupled Plasma/Mass Spectrometry A Performance Evaluation: Single-Laboratory Validation, First Action 2015.06.

A method for determination of 12 minerals and trace elements (Na, Mg, P, K, Ca, Cr, Mn, Fe, Cu, Zn, Se, and Mo) in infant formula and adult/ pediatric nutritional formula was developed and evaluated in a single-laboratory validation. Some additional reproducibility data were obtained from a small interlaboratory study. The method involves microwave digestion of the sample followed by inductively coupled plasma/MS and uses Ge and Te as internal standards. The method is an extension of Official Method(SM) 2011.19 and was compared to AOAC Standard Method Performance Requirements (SMPRs®) 2011.009 and 2014.004 developed by the AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Repeatability precision for the 12 elements in 11 SPIFAN matrixes and National Institute of Standards and Technology Standard Reference Material (SRM) 1849a was <5%, meeting the SMPR criterion for repeatability. Intermediate reproducibility (8 days, two analysts, two instruments) in the 11 SPIFAN matrixes was <5% for nine (Na, Mg, P, K, Mn, Fe, Cu, Zn, Se) of the 12 elements in all 11 matrixes. The mean reproducibility across 6-7 laboratories and seven SPIFAN matrixes ranged from 2.5% for Cu to 7.1% for P. Recovery from spiked matrixes varied from 90.1 to 109%, and accuracy of determination using SRM 1849a ranged from 96.2 to 107.7%, meeting the requirement of 90-110% recovery/accuracy.

SAS molecular tests Escherichia coli O157 detection kit. Performance tested method 031203.

The SAS Molecular tests Escherichia coli O157 Detection method, a loop-mediated isothermal amplification method, performed as well as or better than the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, bagged mixed lettuce, and fresh spinach. Ground beef (30% fat, 25 g test portion) was validated for 7-8 h enrichment, leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was also shown to be acceptable under conditions of co-enrichment with Salmonella. Thus, after a short co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. The SAS Molecular tests Salmonella Detection Kit was validated using the same test portions as for the SAS Molecular tests E. coli O157 Detection Kit and those results are presented in a separate report. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 E. coli 0157 strains, including H7 and non-motile strains, and 30 non-E. coli O157 strains examined. Finally, the method was shown to be robust when variations to DNA extract hold time and DNA volume were varied. The method comparison and robustness data suggest a full 7 h enrichment time should be used for 25 g ground beef test portions.

SAS molecular tests Salmonella detection kit. Performance tested method 021202.

The SAS Molecular tests Salmonella Detection method, a Loop-mediated Isothermal Amplification method, performed as well as or better than the U.S. Department of Agriculture-Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, ground turkey, chicken carcass rinses, bagged mixed lettuce, and fresh spinach. The ground beef (30% fat, 25 g test portion), poultry matrixes and leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was shown to be acceptable under conditions of co-enrichment with Escherichia coli 0157. Thus, after a short 6-7 h co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 100 Salmonella serovars and 30 non-Salmonella species examined. The method was shown to be robust when enrichment time, DNA extract hold time, and DNA volume were varied.

Determination and confirmation of nicarbazin, measured as 4,4-dinitrocarbanilide (DNC), in chicken tissues by liquid chromatography with tandem mass spectrometry: First Action 2013.07.

A single-laboratory validation (SLV) study was conducted on an LC/MS/MS method for the determination and confirmation of nicarbazin, expressed as 4,4-dinitrocarbanilide (DNC), in chicken tissues, including liver, kidney, muscle, skin with adhering fat, and eggs. Linearity was demonstrated with DNC standard curve solutions using a weighted (1/x) regression and confirmed with matrix-matched standards. Intertrial repeatability precision (relative standard deviation of repeatability; RSD(r) was from 2.5 to 11.3%, as determined in fortified tissues. The precision was verified with incurred tissue, and varied from 0.53 to 2.5%. Average recoveries ranged from 82% in egg to 98% in kidney. Although the average recoveries across all concentrations were within the acceptable range, the method was improved with the inclusion of an internal standard and the use of matrix-matched standards. Accuracy for the improved method in chicken liver varied from 93 to 99% across all concentrations (100-8000 ng/g) compared to recoveries below 80% at concentrations, between 100-400 ng/g in chicken liver for the original method. The limit of detection was estimated to be less than 3.0 ng/g in all tissue types, and the limit of quantitation was validated at 20 ng/g. Based on confirmatory ion ratios and peak retention times, the false-negative rate was estimated as 0.00% (95% confidence limits 0.00, 0.74%) from 484 fortified samples and 12 incurred residue samples analyzed using the U.S. and EU confirmation criteria. Small variations to the method parameters, with the exception of injection volume, did not have a significant effect on recoveries. Stability was determined for fortified tissues, extracts, and standard curve solutions. The data collected in this study satisfy the requirements of SLV studies established by the AOAC Stakeholder Panel for Veterinary Drug Residue and the method was awarded First Action Official Method status by the Expert Review Panel for Veterinary Drug Residues on May 7, 2013.

Determination and confirmation of narasin and monensin in chicken, swine, and bovine tissues by LC/MS/MS: Final Action 2011.24.

A multilaboratory study was conducted to validate the reproducibility of AOAC Official Method 2011.24 for determination of narasin and monensin in chicken, swine, and bovine tissues. This study was intended to satisfy requirements for Final Action status through the AOAC Expert Review Panel process. Ten laboratories participated in the study, analyzing blind duplicates of five incurred residue materials for each analyte. After removal of invalid data sets, the method reproducibility (RSDR 12.8-60.6%, HorRat 0.45-1.47) was within AOAC acceptance criteria. The method was awarded Final Action status by the Official Methods Board on October 4, 2012.

Determination and confirmation of parent and total ractopamine in bovine, swine, and turkey tissues by liquid chromatography with tandem mass spectrometry: Final Action 2011.23.

A multilaboratory study of AOAC Official Method 2011.23 was performed to satisfy requirements for Final Action status through the AOAC expert review panel process. The study included nine collaborating laboratories from the United States, Canada, Brazil, and The Netherlands. Five incurred residue materials (bovine muscle, bovine liver, swine muscle, swine liver, and turkey muscle) were analyzed by each laboratory as blind duplicates for parent and total ractopamine content. After removal of invalid data, the parent and total ractopamine methods demonstrated acceptable reproducibility (RSDR 11.4-42.4%, HorRatR 0.34-2.01) based on AOAC criteria. The method was awarded Final Action status by the Official Methods Board on October 4, 2012.

RAZOR EX Anthrax Air Detection System for detection of Bacillus anthracis spores from aerosol collection samples: collaborative study.

The RAZOR EX Anthrax Air Detection System was validated in a collaborative study for the detection of Bacillus anthracis in aerosol collection buffer. Phosphate-buffered saline was charged with 1 mg/mL standardized dust to simulate an authentic aerosol collection sample. The dust-charged buffer was spiked with either B. anthracis Ames at 2000 spores/mL or Bacillus cereus at 20 000 spores/mL. Twelve collaborators participated in the study, with four collaborators at each of three sites. Each collaborator tested 12 replicates of B. anthracis in dust-charged buffer and 12 replicates of B. cereus in dust-charged buffer. All samples sets were randomized and blind-coded. All collaborators produced valid data sets (no collaborators displayed systematic errors) and there was only one invalid data point. After unblinding, the analysis revealed a cross-collaborator probability of detection (CPOD) of 1.00 (144 positive results from 144 replicates, 95% confidence interval 0.975-1.00) for the B. anthracis samples and a CPOD of 0.00 (0 positive results from 143 replicates, 95% confidence interval 0.00-0.0262) for the B. cereus samples. These data meet the requirements of AOAC Standard Method Performance Requirement 2010.003, developed by the Stakeholder Panel on Agent Detection Assays.

Determination and confirmation of parent and total ractopamine in bovine, swine, and turkey tissues by liquid chromatography with tandem mass spectrometry: First Action 2011.23.

A candidate method selected by the AOAC Expert Review Panel (ERP) for Ractopamine for determination and confirmation of parent and total ractopamine by LC/MS/MS was validated in a single laboratory for bovine, swine, and turkey tissues. The candidate method utilizes methanol extraction of the tissues, followed by an optional enzymatic hydrolysis for determination of total (parent plus conjugate) ractopamine. A mixed-mode cation exchange SPE cartridge is used to purify the initial extract before LC/MS/MS. Matrix-matched standards and a ractopamine-d6 internal standard are used for quantification of parent and total ractopamine in unknown samples. Validation data demonstrated that mean intertrial recoveries for ractopamine across all concentrations tested ranged from 79.7 to 102.2% for parent ractopamine and from 79.0 to 100.0% when a hydrolysis step was included. Intertrial repeatability precision ranged from 2.44 to 11.1% for parent ractopamine and 4.97 to 15.0% with hydrolysis. Estimated LOD values were below 0.1 ng/g and LOQ values were validated at 0.25x the maximum residue limits. The data satisfy the requirements of the AOAC Stakeholder Panel for Veterinary Drug Residue Methods for single laboratory validation studies. The method was awarded Official Methods of Analysis First Action 2011.23 by the AOAC ERP on Veterinary Drug Residues.