New analytical methods for the determination of free asparagine Asn , which is a precursor of acrylamide, in grains were developed using LC-MS and LC-MS/MS. Asn was extracted from a sample with 5 w/v aqueous trichloroacetic acid solution, appropriately diluted with 0.1 v/v formic acid solution, and then analyzed by LC-MS or LC-MS/MS. HPLC separation was performed by isocratic elution on a Penta Fluoro Phenyl PFP column using 0.1 v/v formic acid and acetonitrile mixture as the mobile phase. The calibration curve was linear in the range of 0.005-0.1 μ g/mL. The mean recoveries from potato starch, non-glutinous rice flour and whole wheat flour ranged from 95.4 to 100.9 , repeatability RSD ranged from 0.9 to 6.0 , and within-laboratory reproducibility RSDwr ranged from 2.8 to 7.1 . Limits of quantitation LOQs were 7 mg/kg for potato starch, and 5 mg/kg for non-glutinous rice flour. In addition, an inter-laboratory study was performed in 10 laboratories using 5 kinds of grains non-glutinous brown rice flour, corn flour, strong flour, whole wheat flour, and whole rye flour , which naturally contained free asparagine. The HORRAT R values ranged from 0.4 to 1.0. These results are within the range of the procedural manual of the Codex Alimentarius Commission, confirming the effectiveness of the developed procedures.
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