AOAC SPIFAN Chlorate-01 Reviewer Form (July 2022)

AOAC Stakeholder Program on Infant Formula and Adult Nutritionals

CHLORATES/PERCHLORATES REVIEWER FORMS (CHLORATE-01)

Tuesday, July 26, 2022

AOAC INTERNATIONAL 2275 Research Blvd., Suite 300 Rockville, MD, 20850 USA

dboyd@aoac.org 301.924.7077 x126

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate-01 (Resubmission)

Title: Method for the Determination of Chlorate and Perchlorate in Baby Food, Baby Food Ingredients, Infant Formula, and Infant Formula Ingredients by Liquid Chromatography – Tandem Mass Spectrometry Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM Reviewer Name: Reviewer 1 Please Provide a Brief Summary of the Resubmitted Method: Samples are extracted with acetonitrile and 2% formic acid in water then purified with a weak anion exchange SPE. The extracts were analyzed by LC-MS/MS in multiple reaction monitoring (MRM) by electrospray ionization (ESI) in negative ion mode. Quantification is performed by isotopic dilution. Was Method Scope/Applicability Updated? The authors covered all matrices included in the SMPR (LOQ was not obtained for soy protein isolate and plant based IF due to lack of blank matrix) and they analyzed a lot of samples with good results. • Is not clear if MS BF1 means LOQ level. But chlorate chromatograms are not very good. • Is not clear if MS Meat 1 means LOQ level. But chlorate chromatograms are not correct. • Perchlorate chromatograms for some samples seems to have interferences in some transitions • Different integrations in different transitions and different from calibration curve was observed Looking at table 10, we can see different LOQ for different samples. But: • Expression of results (as stated in manuscript) for non-detected amount must be expressed as <0,005 mg/kg. Means this that 0,005 mg/kg is the reporting level that is equal to LOQ? So, the LOQ are the ones in table 10 or the LOQ is 0,005 mg/kg for all matrices? • If the LOQ are the ones in table 10, the authors did not validate at these limits, and these limits are not obtained as stated in SMPR, they are calculated. • In chromatograms presented for the LOQ level, some chlorate signals are very low, lower than S/N = 10 for example MS Meet1 • LOQ was not obtained for soy protein isolate and plant based IF due to lack of blank matrix The HPLC-MS/MS analytical sequence is not in agreement with SANTE guide: the method injects calibration curve at the beginning of the sequence, and then, only one level of calibration curve is Regarding chromatograms: • Infant Formula’s chlorate LOQ chromatograms show interferences.

1

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

reinjected after every 7-10 samples. There are not specifications for the bracketing injections of the same calibration standard. The authors mentioned that no reference materials were available, so, any of the reference materials recommended in the SMPR were analyzed. Regarding Table 9: • How do the authors obtain the incurred concentrations of the samples? Some values are extremely low, looking at the chromatograms, is clear that these values are part of the noise. Results are expressed with a lot of significant figures. • For WPC the lower level used in validation is 0,05 mg/kg. But depending on the amount of WPC added to finished product, the LOQ must be <0,01 mg/kg. Has the resubmitted method met the requirements outlined as per the ERP recommendation/comment(s)? Most of ERP’s requirements were included, but not all of them. Method was not improved (authors stated that gradient elution was not recommended for the column type they are using, and therefore, this was not investigated). No information about results calculated with calibration that is forced through

zero & not forced through zero. Please provide documentation:

All information is included in Manuscript.

Provide General comments: The authors covered all matrices included in the SMPR and they analyzed a lot of samples with good results. Most of ERP’s requirements were included.

Some LOQ are not correct, and interferences are still present.

Final Recommendation: Very promising method, but I still not recommend it for First Action before discussions about LOQ and chromatograms interferences.

2

Reviewer 2 RSD Calcs (July 2022)

IncurredCon c.(mg/kg)

SpikedConc. (mg/kg) Recovery(%)

Matrix Category

Matrix

Compound

Beef/Rice/Vege Baby Food

Baby food

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

0.00024 0.00024 0.00024 0.00024 0.00024 0.00024 0.00024 0.00024 0.00024 0.00024 0.00120 0.00120 0.00120 0.00120 0.00120 0.00120 0.00120 0.00120 0.00120 0.00120 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077 0.00077

0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050

93 95

112 105

90 98 97 91 92 92

102

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

118 118

92

119

97 99 91 88 90

Baby food ingredients

Cereal Ingredients

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

101 107 103

99 97 99 94 98 95 94 99 98

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

102 104

81 95 98

100

Page 1 of 14

Reviewer 2 RSD Calcs (July 2022)

Perchlorate Perchlorate

0.00077 0.00077 0.00121 0.00121 0.00121 0.00121 0.00121 0.00121 0.00121 0.00056 0.00056 0.00056 0.00056 0.00056 0.00056 0.00056 0.00159 0.00159 0.00159 0.00159 0.00159 0.00075 0.00075 0.00075 0.00075 0.00075 0.01239 0.01239 0.01239 0.01239 0.01239 0.01239 0.01239 0.01239 0.01239 0.01239 0.00305 0.00305 0.00305 0.00305

0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.500 0.500 0.500 0.500 0.500 0.050 0.050 0.050 0.050

97 96 97 93 85 83 92 88

Kiwifruit

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

106

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

115 110 105 109 101 112 108 105 108 107 99 98

Meat Ingredients

Chlorate Chlorate Chlorate Chlorate Chlorate

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

97

104

89

105

94

Infant/adult formula ingredients

Whey Protein Concentrate

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

92 91 95 91 98 97 95 99 96 89 93 92 95

102

Perchlorate Perchlorate Perchlorate Perchlorate

Page 2 of 14

Reviewer 2 RSD Calcs (July 2022)

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

0.00305 0.00305 0.00305 0.00305 0.00305 0.00305 0.01274 0.01274 0.01274 0.01274 0.01274 0.01274 0.01274 0.00228 0.00228 0.00228 0.00228 0.00228 0.00228 0.00228 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.01467 0.00086 0.00086 0.00086 0.00086 0.00086 0.00086 0.00086 0.00086 0.00086

0.050 0.500 0.500 0.500 0.500 0.500 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.050 0.050

97 96 98

105 101 104 111 109 115 104 105 82

Whole Milk Powder

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

81 84 92 82 87 99 92 99 98 99 93 91 97 93

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

Lactose

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

107

112

101

95

106 107

99

107

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

97 88 87 89 82 86 89 94

105

Page 3 of 14

Reviewer 2 RSD Calcs (July 2022)

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

0.00086 0.00086 0.00086 0.00086 0.00086 0.00148 0.00148 0.00148 0.00148 0.00148 0.00196 0.00196 0.00196 0.00196 0.00196 0.00135 0.00135 0.00135 0.00135 0.00135 0.00123 0.00123 0.00123 0.00123 0.00123 0.00090 0.00090 0.00090 0.00090 0.00090 0.00090 0.00090 0.00090 0.00090 0.00090 0.00091 0.00091 0.00091 0.00091 0.00091 0.00091 0.00091 0.00091

0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050

90 89 84 93 97 97 93 95 93 95 81 87 86 98 84 91 90 89 94 92 85 89 88 91 98 95 89

Maltodextrin

Chlorate Chlorate Chlorate Chlorate Chlorate

109

103

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

166

Oligosaccharides

Chlorate Chlorate Chlorate Chlorate Chlorate

132 113

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

116

Oil/Fat

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

103 113

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

105 115 103 112 117 101 103 97

Page 4 of 14

Reviewer 2 RSD Calcs (July 2022)

Perchlorate Perchlorate

0.00091 0.00091 0.01670 0.01670 0.01670 0.01670 0.01670 0.01670 0.01670 0.00114 0.00114 0.00114 0.00114 0.00114 0.00114 0.00114 0.00382 0.00382 0.00382 0.00382 0.00382 0.00382 0.00382 0.00382 0.00382 0.00382 0.01834 0.01834 0.01834 0.01834 0.01834 0.01834 0.01834 0.01834 0.01834 0.01834 0.00164 0.00164 0.00164 0.00164 0.00164 0.00164

0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050

106 101

Liquid Milk

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

93 96 92 88 88 96

107

106

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

107 103 101

86 87 95

Sheep Milk

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

173 103

88

101

99 94 96 95 89

100 111 115 118

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

81

120

91 93 84 82 93 93 97 88 91 91

Mineral and vitamin based premixes

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

117

Page 5 of 14

Reviewer 2 RSD Calcs (July 2022)

Chlorate Chlorate Chlorate Chlorate

0.00164 0.00164 0.00164 0.00164 0.00082 0.00082 0.00082 0.00082 0.00082 0.00082 0.00082 0.00082 0.00082 0.00082 0.24738 0.24738 0.24738 0.24738 0.24738 0.24738 0.24738 0.24738 0.24738 0.24738 0.00891 0.00891 0.00891 0.00891 0.00891 0.00891 0.00891 0.00891 0.00891 0.00891 0.02240 0.02240 0.02240 0.02240 0.02240 0.02240 0.02240

0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.800 0.800 0.800 0.800 0.800 1.300 1.300 1.300 1.300 1.300 0.800 0.800 0.800 0.800 0.800 1.300 1.300 1.300 1.300 1.300 0.005 0.005 0.005 0.005 0.005 0.005 0.005

95 94

104

94

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

109 179 108 105 89

92 93

109

97

108

Soy Protein Isolate

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

96 99 97 99 92 95 96 93 96 99

100

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

102 100

99

102

99 97

102

98

102

Infant/adult formulas

Infant Formula

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

107 102 107 104 119 109 95

Page 6 of 14

Reviewer 2 RSD Calcs (July 2022)

Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate Chlorate

0.02240 0.02240 0.02240 0.02240 0.02240 0.02240 0.02240 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.00268 0.13035 0.13035 0.13035 0.13035 0.13035 0.00428 0.00428 0.00428 0.00428 0.00428

0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.005 0.005 0.005 0.005 0.005 0.005 0.005 0.050 0.050 0.050 0.050 0.050 0.050 0.050 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500 0.500

102 114 110 112 114 106

95 98 87 92

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

110 113 153 107

91 96 92 91 96 84

110

Plant based IF

Chlorate Chlorate Chlorate Chlorate Chlorate

100

97

100 98 98 97 91 100 100 103

Perchlorate Perchlorate Perchlorate Perchlorate Perchlorate

Page 7 of 14

Reviewer 2 RSD Calcs (July 2022)

Recovered conc (mg/kg)

RSDr (per conc)

Average Std Dev

RSD (overall)

0.00465 0.00495 0.00045962

9.29

7.00

0.00475 0.0056 0.00525 0.0045 0.049

0.048 0.00226385

4.72

0.051 0.0485 0.0455 0.046 0.0046 0.00539 0.00072146

13.39

9.24

0.0059 0.0059 0.0046 0.00595 0.0485 0.0495 0.0455

0.0465 0.00237171

5.10

0.044 0.045

0.00505 0.00507 0.00019235

3.79

3.12

0.00535 0.00515 0.00495 0.00485 0.0495

0.048 0.0011726

2.44

0.047 0.049 0.0475 0.047 0.00495 0.00484 0.00045744

9.45

5.72

0.0049 0.0051 0.0052 0.00405 0.0475

0.0486 0.00096177

1.98

0.049 0.05

Page 8 of 14

Reviewer 2 RSD Calcs (July 2022)

0.0485 0.048 0.00485 0.00465 0.00425 0.0053 0.00415

0.0046 0.00039158

8.51

8.51

0.0046 0.0044 0.00575 0.00536429 0.00029257 0.0055 0.00525 0.00545 0.00505 0.0056 0.00495 0.0049 0.00526 0.00021036 0.0054 0.00525 0.0054 0.00535 0.00485 0.00489 0.00033801

5.45

5.45

4.00

4.00

6.91

6.91

0.0052 0.00445 0.00525 0.0047

0.046 0.0455 0.0475 0.0455 0.475 0.51 0.495 0.48 0.0445 0.0465 0.046 0.0475 0.049 0.485

0.0467 0.0015248

3.27

3.05

0.489 0.01387444

2.84

0.0466 0.00151658

3.25

3.53

Page 9 of 14

Reviewer 2 RSD Calcs (July 2022)

0.0485 0.48

0.504 0.01917029

3.80

0.49 0.525 0.505 0.52 0.0041 0.00505 0.00069101

13.68

13.68

0.00555 0.00545 0.00575 0.0052

0.00525 0.00405 0.0042 0.00453571 0.00033877

7.47

7.47

0.0046 0.0041

0.00435 0.00495 0.0046 0.00495 0.0049 0.00497857 0.00037401

7.51

6.62

0.00535 0.00495 0.00465 0.0056

0.00455 0.00485 0.0465 0.05057143 0.00289293

5.72

0.0505 0.0475 0.053

0.0535 0.0495 0.0535 0.00485 0.00452857 0.00039036 0.0044 0.00435 0.00445 0.0041 0.00525 0.0043 0.0445 0.04542857 0.00211007

8.62

6.63

4.64

0.047

Page 10 of 14

Reviewer 2 RSD Calcs (July 2022)

0.045 0.0445 0.042

0.0465 0.0485 0.00485 0.00491 0.00039749

8.10

8.10

0.00465 0.00545 0.00445 0.00515 0.00475

0.0053 0.0017022

32.12

32.12

0.00465 0.00475 0.00405 0.0083 0.00435 0.00516 0.00097173

18.83

18.83

0.0043 0.0049 0.0066 0.00565 0.0042

0.0047 0.00062948

13.39

13.39

0.00455 0.0058 0.0045 0.00445 0.0047 0.00487 0.00054153

11.12

7.84

0.0046 0.00425 0.00515 0.00565 0.0445

0.0461 0.00210357

4.56

0.044 0.0455 0.049 0.0475 0.00525 0.00552 0.00030741

5.57

4.40

0.00575 0.00515 0.0056 0.00585 0.0485 0.0505 0.0515

0.0508 0.00164317

3.23

Page 11 of 14

Reviewer 2 RSD Calcs (July 2022)

0.053 0.0505 0.00465 0.00478571 0.00039446

8.24

8.24

0.0048 0.00535

0.0046 0.0044 0.0053 0.0044 0.0048 0.00482143 0.00039566

8.21

8.21

0.00535 0.00515 0.00505 0.0043

0.00435 0.00475 0.00865 0.00564 0.00170748

30.27

17.23

0.00515 0.0044 0.00505 0.00495 0.047

0.0474 0.00198116

4.18

0.048 0.0475 0.0445 0.05 0.00555 0.00545 0.00080078

14.69

10.30

0.00575 0.0059 0.00405 0.006 0.0455 0.0465 0.042 0.041 0.0465 0.00485 0.0044 0.00585 0.00455 0.0455

0.0443 0.00261247

5.90

0.00465 0.00486 0.00057706

11.87

8.52

0.0478 0.00246475

5.16

Page 12 of 14

Reviewer 2 RSD Calcs (July 2022)

0.0475 0.047 0.052 0.047 0.00545 0.00895 0.00445 0.0054 0.00525 0.046 0.0465 0.0545 0.0485

0.0059 0.00175214

29.70

18.94

0.0499 0.0040835

8.18

0.054 0.768 0.792 0.776 0.792 0.736 1.235 1.248 1.3 1.209 1.248 0.792 0.816 0.8 0.792 0.816 1.287 1.261 1.326 1.274 1.326

0.7728 0.02304778

2.98

2.82

1.248 0.03314363

2.66

0.8032 0.0121326

1.51

1.91

1.2948 0.02992825

2.31

0.00535 0.00530714 0.00036564

6.89

6.73

0.0051 0.00535 0.0052 0.00475 0.00595 0.00545

Page 13 of 14

Reviewer 2 RSD Calcs (July 2022)

0.051 0.05378571 0.00353385 0.057 0.055 0.056 0.057 0.053 0.0475 0.0049 0.00542857 0.00108967

6.57

20.07

14.29

0.00435 0.0046 0.0055

0.00565 0.00765 0.00535 0.0455 0.04714286 0.00400743

8.50

0.048 0.046 0.0455

0.055 0.048 0.042 0.5 0.485 0.5

0.493 0.0067082

1.36

1.36

0.49 0.49 0.5

0.491 0.02274863

4.63

4.63

0.485 0.455 0.5 0.515

Page 14 of 14

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Chlorate-01 (Resubmission)

Title: Method for the Determination of Chlorate and Perchlorate in Baby Food, Baby Food Ingredients, Infant Formula and Infant Formula Ingredients by Liquid Chromatography – Tandem Mass Spectrometry: Single Laboratory Validation Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM Reviewer Name: Reviewer 3 Please Provide a Brief Summary of the Resubmitted Method: The method is based on an extraction with acetonitrile and water acidified with 2% formic acid and a subsequent clean-up based on SPE (weak anion exchange) and determination by liquid chromatography coupled to isotope dilution tandem mass spectrometry (LC-MS/MS).

Was Method Scope/Applicability Updated?

It was partially (please see General comments below).

Has the resubmitted method met the requirements outlined as per the ERP recommendation/comment(s)?

As in the previous Re-submission, It has partially:

- Validation experimental design and results of the study are better described although they do not fully meet the SMPR requirements yet (please, see General comments below). - Calculations have been significantly simplified, although they still need some more clarifications (please, see General comments below).

Please provide documentation:

Provide General comments: 1.- Calculations

1

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

- In equation c, please indicate what is m and its units. - Equations c and d could be combined to simplify the calculation procedure

2.- Validation - LOQ is still estimated as 3.14 x 3 x SD of either 5 or 7 determinations at 0.005 mg/kg. According to the SMPR the LOQ should determined as the lowest spiking level that meets recovery and repeatability requirements”. Meaning, that in this example, it would be 0.005 mg/kg (provided that the accuracy and precision are within the SMPR criteria). - In the case of baby food (finished products) estimation was done pulling down 5 determinations including vegetables, cereals and meat-based baby food, meaning that there are no independent data for each of those matrix subcategories. This was also the case for the estimation of the precision of the method in this matrix category. - The experimental design for the estimation of the precision of the method should be detailed as well as the calculations applied for its estimation. Where the results obtained in different analytical batches? According to the SMPR: Repeatability.—Variation arising when all efforts are made to keep conditions constant Reproducibility.—Standard deviation or relative standard deviation calculated from among-laboratory data. 5 determinations seem to be less than enough for estimation of the two components of the precision simultaneously (in some cases there were even 4 because of the exclusion of 1 outlier, i.e. Perchlorate (maltodextrin, minerals and vitamin premixes) Chlorate (sheep milk), because those replicates results were either obtained on different analytical batches (for reproducibility estimation) or combined in a minimum of 2xrep x 2 batches + 1 rep which gives not enough degree of freedom for repeatability estimation. - Table 11 indicates the precision for chlorate and perchlorate (both RSDr and RSD ip). As indicated in previous method revision, again those figures are obtained after pooling down all the results (138 for chlorate, and 136 for perchlorate). This pool includes results at different concentration levels, which may hide a lower performance of the method at low concertation level, and in any case does not give the figure at the LOQ concentration levels as required in the SMPR. Additionally, the number of determinations for each matrix can vary from 4 to 7, which gives more significance to some commodities that to others. - Table 9 includes recovery of individual determinations and not individual precision samples as indicated in its legend. - Table 12 is referred in the manuscript as including the values for Intermediate Reproducibility estimation, however it includes only values on accuracy (recoveries), and it seems to be a summary of table 10 (recovery of individual determination). For clarification, please, specify the formulae used for estimation of for RSDip and RSDr?

2

FOR EXPERT REVIEW PANEL USE ONLY NOT FOR DISTRIBUTION

Final Recommendation: Performance of the method was not proved to meet the requirements defined in the SMPR, especially for accuracy on cereal-, fruits/vegetables- and meet-based baby food, and generally on precision estimation across matrix subcategories specified on the SMPR, therefore the method can not be recommended further for first action.

3