AOAC SPIFAN Nutrients ERP Reviewer Forms (December 7, 2022)

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Cons/Weaknesses • 13 C labeled 2-AB Internal standard must be synthesized as it is not commercially available. • Method requires instrumentation that may not be available at laboratories. • Method performance requirements (based on SMPR) were not met for all HMO. To my understanding, the acceptance criteria were mainly based on the 2’FL and LNnT SMPRs.

Supporting Data • General Comment:

Not sufficient supporting data were provided. The method includes several tables where results are presented. However, validation raw-data that could be used to re-calculate and double-check the data were not provided.

Method Optimization:

Not provided.

• Performance Characteristics: In paragraph 2.3 of the method only the draft SMPR for 2’FL and LNnT are mentioned, although for all discussed HMOs are SMPRs published and are available. To my understanding, the method has been developed based on the 2’FL and LNnT. The Method Performance Criteria of the other HMOs should be considered. Analytical Range: No information provided in the text. Should be clearly indicated and discussed based on the SMPRs. Provided was only the calibration range (0.1 – 75 µ g/mL) LOQ: Reported is 2.5 HMO mg/100g milk with set criterion of 4mg/100g milk. The LOQ requirement is not met for 6’SL, 3’SL and DFL. Information about the required LOQ for each HMO is provided in the published SMPRs. Accuracy/Recovery: The reported recovery was between 90.1 – 97.2 %, 96.5-103% and 98.5-104% for the low, middle and high level QC A samples. However, regarding the low levels in HMO, the recovery acceptance criterion (85-110 %) was based on the concentration range of 2.5-20mg/100g. For a number of HMOs the SMPR request a lowest limit that is lower than 2.5g/100g. It is advisable to consult the published SMPR.

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