Determination of Bromoform in Seaweed, Oil, and Animal Feed by GC-MS/MS: AOAC Official Method 2026.01, First Action

BACKGROUND: Bromoform is the bioactive compound present in Asparagopsis seaweed responsible for methane reduction in ruminants. Although several analytical methods are available, no AOAC Official Method exists. OBJECTIVE: A study was performed to validate a GC-MS/MS-based method for determination of bromoform in fresh and freeze-dried seaweed, canola oil containing bromoform (oil herein), and liquid and dry cattle feed. METHODS: The method was evaluated according to AOAC guidelines for calibration curve validity, selectivity, extraction protocol robustness, and matrix and extract stability. Repeatability, recovery, and limits of detection and quantification were also determined for each matrix. RESULTS: The calibration curve was linear over the range 0.000124-0.020 µg/mL (R2= 0.999), and residual analysis demonstrated suitability of the linear model. There was no interference from compounds similar to bromoform (dibromomethane, dichloromethane, dibromochloromethane, and dibromoacetic acid). The analyte was identified by comparison of retention time and ion abundance profiles between calibration standards and unknown samples. The LOQs were 0.057 mg/g, 0.423 mg/g, 0.033 mg/g, 0.022 mg/g, and 0.041 mg/g for fresh seaweed, freeze-dried seaweed, oil, liquid feed, and dry feed, respectively. Bromoform recovery among matrixes across multiple fortified concentration levels was 93-104%, and repeatability was 0.75-5.4%. The method was robust to minor changes in extraction conditions. This method was demonstrated to be accurate and precise with or without the inclusion of an internal standard. CONCLUSION: The GC-MS/MS method for bromoform determination presented in this report was shown to be fit for purpose and has been accepted as a First Action AOAC Official Method SM. HIGHLIGHTS: This method is the first bromoform analysis method to successfully complete the AOAC validation process and will serve to standardize the determination of bromoform in feed ingredients and finished supplements. The adoption of this method is timely given the recent interest in methane reduction in cattle using feed fortified with bromoform.