July 2019 Sugar ERP - Review Book

Sugar Method Re-submission Review Form

Submission Date

2019-07-01 09:08:59

Name

roberto molteni

Email

r.molteni@politicheagricole.it

Organization

Italian Ministry of Agriculture

Candidate Method Number:

SUG-04

Has the resubmitted method met the requirements outlined on August 29, 2018?

Yes

Please provide justification for your answer. If specific requirements were made in the August 29, 2018 meeting, please explain how those requirements have been met or not met.

- Test portion has been addressed: normal procedure is 2 g of sample but for samples that contain more than 50% of any individual sugar, weight is about 1 g.

- Additional reference materials have been included in the submission. Finally, the reference materials are the following (in brackets, reference value of the sugar g/100g): NIST 1849 Infant/adult nutritional formula Lactose (47.6) NIST SRM 1869 Infant Formula Glucose(2.00) Sucrose(27.88) Lactose(0.52) Maltose(2.64) NIST SRM 3233 Fortified Breakfast Cereal Fructose(0.81) Glucose(1.04) Sucrose(13.42) Maltose(0.46) NIST SRM 3282 Low Calorie Cranberry Juice Fructose(2.08) Glucose(0.85) MUVA CA-0904 Sodium Caseinate Lactose(0.023)

MUVA 2310 UHT Low-Lactose Milk Galactose(2.2) Glucose(2.25) Lactose(0.212) MUVA 2311 UHT Lactose-Free Milk Galactose(2.4) Glucose(2.4) Lactose(0.007).

- Potential interferences, specifically isobaric interferences, have been addressed showing one example of chromatogram and listing potential interfering isobaric compounds. However, the only one chromatogram does not seem a chromatogram of sample.

- System suitability and analytical QC, with blanks and check standards, are not explained in details: e.g. Inject at X replicates of the check standards at the beginning and X check standard after every X sample as well as at the end of each sequence.

- Matrix coverage support the scope of the method. Finally, the matrix are the following (in brackets, average value of the sugar g/100g): Ground Corn Meal Fructose(0.142) Glucose(0.181) Sucrose(1.417)

Dry Pet Food Fructose(0.114) Glucose(0.064) Sucrose(0.375) Maltose(0.078) Granola Bar Fructose(1.835) Glucose(2.475) Sucrose(21.272) Maltose(3.275) Cheese Spread Fructose(0.909) Galactose(0.137) Glucose(1.045) Lactose(6.820) and several different reference materials.

- Temperature and % ethanol of extraction have been addressed with the Extraction Robustness Study, showing that 25% alcohol in water at 60 °C efficiently suppressed enzyme activity. However, it would have been better showing overlay chromatograms of sugars: 1) yoghurt; 2) yoghurt spiked with 0.1%, 2%, and 20% levels of sugars of interest.

- Representative chromatograms of samples are not provided.

Overall, I would like to add some points:

1) Reproducibility is mentioned in the method, however the precision calculated fits with definition of the repeatability (within laboratory during short time) or intermediate precision (ISO 5725: 1994) if run not simultaneously, i.e., on different days, with different calibration curves, with different instruments, by different analysts, etc.

2) The RSD values estimated in the table “Reproducibility Results: Experiment” are of the same magnitudes of the RSD values in the table “Replicate Injection Results”. It is unusual, because experiments, with all the sample preparation, is less precise than injection. If “Experiment” means again only injection, the estimated RSD is not the precision of the method. 3) Regarding the LOQ determination, it is written: “since the mathematically determined LOQ is far smaller than the theoretical LOQ of 0.005%. It is concluded that the method can reliably quantify lactose (and likely other five sugars) at as low as 0.005%”. It should be true, however representative chromatograms of low concentration samples could be show it.

4) The Internal Standard is added during the dilution step, just before filtration through 0.2 µm nylon filter into autosampler vials for LC analysis. Why is not added at the begin of the sample preparation to improve the analysis?