AOAC SPIFAN Chlorates Reviewer Form (December 2021)

AOAC Stakeholder Program on Infant Formula and Adult Nutritionals (SPIFAN)

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

CHLORATES/PERCHLORATES REVIEWER FORMS (CHLORATE-01)

December 2021 AOAC INTERNATIONAL 2275 Research Blvd., Suite 300 Rockville, MD, 20850 USA

dboyd@aoac.org 301.924.7077 x126

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method____ Chlorate 01__________ Title: Chlorate and Perchlorate in Baby Foods, Infant/Adult Formulas, and Their Ingredients LC-MS/MS Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM Reviewer Name: Reviewer 1 Summary of Method: Samples are spiked with isotopically labelled internal standard, followed by extraction with acetonitrile and 2% formic acid in water. After centrifugation, the supernatant is subjected to a weak anion exchange solid phase extraction, eluted with 5% ammonia in acetonitrile then concentrated. The extract is analyzed by LC-MS/MS in multiple reaction monitoring (MRM) by electrospray ionization (ESI) in negative ion mode. Analytes are reported in terms of the free anion. . Method Scope/Applicability: The method is applicable to the determination of 0.005 to 1.0 mg/kg of chlorate and perchlorate in Dairy Products, Fruit, Vegetables, Grains, and Baby Food. General comments about the method: Is a good method, it is easy to perform and meets the SMPR. Method Clarity: The method is straightforward and is clearly written.

Pros/Strengths: • Straightforward, fast. It uses common reagents. Good results during SLV. Cons/Weaknesses • Bad peak shapes. Supporting Data • General Comment :

Method Optimization: the method was well optimized regarding extraction and MSMS parameters. Chromatography has a good resolution between peaks, but peak shape could be better

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• Performance Characteristics :

Analytical Range: The method states 0.005 to 1.0 mg/kg of chlorate and perchlorate, but the report limit is <0,01 mg/kg LOQ: 0.005 to 1.0 mg/kg of chlorate and perchlorate but they reported non-detected amount as <0.01 mg/kg Accuracy/Recovery: Recovery meets the SMPR. Authors mention “Recoveries determined for 5-26 test materials spiked with 0.005-1.0 mg/kg of chlorate and perchlorate ranged from 97 to 104%. Recoveries are within the 80-131% range with higher recoveries being observed when incurred levels in the test materials were approaching the fortification level.” But this information, is not described in tables. It is necessary to include n=number of samples in tables with SLV parameters data. It is necessary to list each matrix, level of the analytes and number of samples in Table 11. The information in Table 11 only shows the average results, but not all the necessary information. Good results obtained in Interlaboratory Test. No Reference Materials used. Precision (RSD r ): Repeatability meets the SMPR but it is necessary to list each matrix, level of the analytes and number of samples in Table 11. The information of Table 11 only shows the average results, but not all the necessary information. Reproducibility (RSD R ): MLT was not performed at this moment.

• System suitability:

During the SLV the Relative Retention Time (RRT) was controlled but this control is not a requirement in the method. Requirements of the method: Before any batches are analyzed on the LC-MS/MS, it should be ensured that there is baseline separation between the chlorate and perchlorate peaks. A representative matrix prepared in duplicate is included in each analytical batch to act as QC

matrix spike and matrix blank samples. Blank reagents is included in each batch.

1. Is the Validation Study Report in a format acceptable to AOAC? Yes 2. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? Yes 3. Are the figures and tables sufficiently explanatory without the need to refer to the text? No. It is necessary to list each matrix, level of the analytes and number of samples in Table 11. It is necessary to include n=number of samples in the tables with SLV parameters data.

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4. Are all the figures and tables pertinent? Yes 5. Could some be omitted and covered by a simple statement? No 6. Are the references complete and correctly annotated? Authors are using a very old version of SANTE Guide. Commission Decision 2002/657/EC was repealed and substituted for COMMISSION IMPLEMENTING REGULATION (EU) 2021/808 of 22 March 2021.

7. Does the method contain adequate safety precaution reference and/or statements? Yes

Recommendation:

I recommend moving to First Action after including more information.

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

Title: Chlorate and Perchlorate in Baby Foods, Infant/Adult Formulas, and Their Ingredients Author: Adelle K. Nancekivell, Elsa M. Lotz, Lisa A. Graham Reviewer Name: Reviewer 2 Summary of Method: Samples are spiked with isotopically labelled internal standards ( 18 O 3 ), followed by extraction with acetonitrile and 2% formic acid in water. After centrifugation, the supernatant is subjected to a weak anion exchange solid phase extraction, eluted with 5% ammonia in acetonitrile then concentrated. The extract is analysed by LC-MS/MS in multiple reaction monitoring (MRM) by electrospray ionisation (ESI) in negative ion mode. Analytes are reported in terms of the free anion. Quantification is performed against solvent-based calibration standards. Method Scope/Applicability: The method is suitable for the determination of chlorate and perchlorate in dairy products, fruit, vegetables, grains, and baby food. The method is applicable to the determination of 0.005 to 1.0 mg/kg of chlorate and perchlorate. Note that the following have not been included from Table 1 of SMPR 2021.001: finished baby food products (any); meat- and cereal-based baby food ingredients; finished infant formulas (any); an adequate representation of any infant formula ingredient category. Thus, according to the SMPR, additional validation is required on more matrices to meet the requirements. Matrix Category Matrix Sub-Category Minimum Representative Matrices SMPR Met? Additional Validation Data Needed for Baby food Cereal-based Fruit/vegetable-based Meat-based NO All parameters All parameters All parameters

Baby food ingredients Infant/adult formulas

Cereal-based Fruit/vegetable-based Meat-based

NO

LOQ -- All parameters LOQ, RSDr All parameters All Parameters -- All parameters All parameters LOQ, RSDr All parameters All parameters

Milk-based Plant-based

NO

Animal-based milk powdered protein sources

Whole milk powder Whey protein concentrate Soy protein isolate Bovine One additional species

NO

-- LOQ, RSDr, Per Recov*

Plant-based protein sources

NO NO NO NO

Liquid milk

Infant/adult formula ingredients

Fat-based

Oil/fat

Carbohydrate-based

Lactose Maltodextrin Oligosaccharides

Mineral- and vitamin-based (premixes) All parameters *assuming whey protein hydrosylate can substitute for whey protein concentrate. If not, all parameters Any NO

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General comments about the method: Overall, this is a good approach to analysis of chlorate and perchlorate in these matrices. The initial validation presented here is thorough and can be expanded to additional matrices to fulfill the requirements of SMPR 2021.001. Some minor comments or concerns are discussed below around the correction factor, retention suitability parameter, and separation conditions. While the method performs well, the chromatographic peak shape is not great and could be improved. Method Clarity: The method is clearly written and easy to follow. No concerns. Pros/Strengths: • Method utilizes an isotope dilution approach for quantitation to improve precision. • Glassware rinsed thoroughly with methanol prior to use to remove any residual chlorate/perchlorate from the environment. • Demonstrates sufficient sensitivity, recovery, and repeatability on several desirable matrices. Cons/Weaknesses • Chromatographic peak shape could be improved • More details needed about the MRMs used for calculation of concentrations in unknown samples, or how a final result is determined. Supporting Data • General Comment: A lot of data is provided but not adequate to meet the SMPR matrix list. Additionally, data for recovery is provided as average and range without the ability for evaluation of individual outlying data points. Method Optimization: Only sample size is optimized; comparison of 2 g versus 1 g of sample.

• Performance Characteristics:

Analytical Range: Described in Table 9. While not specifically delineated in SMPR 2021.001, the lower end of the linear range described here is 10x greater in the procedural samples when compared to the required LOQ. Given that the following section describes lower LOQs, the linear range can likely be extended to lower levels.

Analyte

Calibration

Concentration Range

Concentration Units

R 2

Slope

Intercept

Chlorate

Solvent Based 0.05 – 5.0

mg/L

0.999 1.000 0.999 1.000

0.7922 0.9075 8.564

0.04403 -0.00516

Procedural

0.05 – 5.0

mg/kg mg/L mg/kg

Perchlorate Solvent Based 0.01 – 1.0

0.0487 0.0969

Procedural

0.01 – 1.0

9.017

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LOQ: Described in Table 10. Calculated using seven replicates fortified at 0.005 mg/kg. LOD defined as SD of replicates x 3.14 (99% confidence, 1 sided t-test). LOQ = 3 x LOD. Two of the product categories (infant/adult formulas and minor ingredients) were not evaluated. In addition, chlorate performance was slightly higher than the LOQ requirement in two product categories (see red below). Matrix Compound LOD (mg/kg) LOQ (mg/kg) SMPR category SMPR Req (mg/kg) Whole Milk Powder Chlorate 0.0014 0.0043 Major IF ingredient ≤0.0 1 Perchlorate 0.0023 0.0055 ≤0.0 1 Whole Milk Chlorate 0.0018 0.0055 Liquid milk ≤0.005 Perchlorate 0.00087 0.0026 ≤0.005 Kiwi Fruit Chlorate 0.0017 0.0051 ≤0.005 Accuracy/Recovery: Described in Tables 11 and 12. Dairy products (4-5) = whey protein hydrolysate (chlorate only), whole milk powder, lactose, whole milk, acid casein. Fruits, vegetables, grains, baby food (26) = apple, banana, nectarine, raspberry, potato, lemon, orange, onion, mushroom, pumpkin, beans, asparagus, avocado, silver beet, cabbage, wheat, hay, spinach, grape, coffee beans, sweet corn powder, lentils, date paste, and fruit and vegetable based baby food, meat based baby food, cereal based baby food) plus 32 replicates of kiwifruit. Average recoveries, save perchlorate in dairy products, are adequate based on this when considering the method uncertainty. Would be nice to review the full data set to determine which matrices/levels are problematic to allow for proper scoping of the method in future. Matrix Compound Fortification (mg/kg) Recovery (%) Recovery Range (%) U (%) SMPR Req (%) Baby food ingredient Perchlorate 0.0010 0.0022 ≤0.005

Dairy products

Chlorate

0.05, 0.2, 1.0 102 0.01, 0.1, 1.0 109

96-121 90-131 80-119 81-127

11 80-120 17 80-120 16 80-120 18 80-120

Perchlorate

Fruit, vegetables, grains, baby food*

Chlorate

0.005, 0.01,* 0.1, 1.0

97

Perchlorate

100

*all samples fortified at 0.01 mg/kg, only kiwifruit fortified at multiple levels

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Additionally, recoveries evaluated using a NIST interlaboratory comparison for infant formulas and ingredients as well as BIPEA standards. Results for one NIST sample (Infant Formula D) and one BIPEA sample (orange) were outside of the required recovery range for chlorate (see below in red).

Assigned/ Consensus Result (mg/kg)

Reported Result (mg/kg)

Recovery (%)

SMPR Req (%) 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120 80-120

ILCP NIST

Sample ID 20-37961-1A

Matrix

Analyte Chlorate

Infant Formula B Infant Formula C Infant Formula D Infant Formula E Infant Formula F

0.11

0.12

88 NA 98 NA

Perchlorate <0.010

0.00062

NIST

20-37961-2A

Chlorate

0.30

0.30

Perchlorate <0.010

0.00045

NIST

20-37961-3A

Chlorate

0.070

0.050

140 NA 109

Perchlorate <0.010

NA 1.4

NIST

20-37961-4A

Chlorate

1.6

Perchlorate 0.026

0.030

87 92 NA 91 95

NIST

20-37961-5A

Chlorate

0.37

0.40

Perchlorate <0.010

0.0058

Chlorate

0.11

0.12

BIPEA

20-102720-1A Cow Milk Powder 20-269201-1A Grape

Perchlorate 0.079

0.083 0.060 0.033 0.040 0.034 0.067

Chlorate

0.072

120

BIPEA

Perchlorate 0.031

94

Chlorate

0.049

123 115 113 107

BIPEA

20-322527-1A Orange

Perchlorate 0.039

Chlorate

0.076

BIPEA

20-102727-1A Spinach

Perchlorate 0.19

0.18

Precision (RSD r ): Described in Table 11. Fortification was at 0.005 mg/kg for seven (7) replicates. This meets the SMPR but was only tested at a single level (LOQ), not at 10x LOQ as required by the SMPR. Matrix Compound RSDr (%) Requirement (%) Dairy products (whole milk powder, whole milk) Chlorate 12 ≤20 Perchlorate 10 ≤20 Kiwifruit Chlorate 7.4 ≤20 Perchlorate 4.3 ≤20

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Reproducibility (RSD R ): Described in Table 11. Dairy products (4-5) = whey protein hydrolysate (chlorate only), whole milk powder, lactose, whole milk, acid casein. Fruits, vegetables, grains, baby food (26) = apple, banana, nectarine, raspberry, potato, lemon, orange, onion, mushroom, pumpkin, beans, asparagus, avocado, silver beet, cabbage, wheat, hay, spinach, grape, coffee beans, sweet corn powder, lentils, date paste, and fruit and vegetable based baby food, meat based baby food, cereal based baby food) plus 32 replicates of kiwifruit. While not required at this stage, the method appears to perform well and will likely be successful through MLT.

Fortification (mg/kg)

RSDR (%)

Requirement (%)

Matrix

Compound

Dairy products

Chlorate

0.05, 0.2, 1.0 5.7 0.01, 0.1, 1.0 8.5

≤2 5 ≤2 5 ≤2 5

Perchlorate

Fruit, vegetables, grains, baby food*

Chlorate

0.005, 0.01,* 0.1, 1.0

8.2 9.2

Perchlorate ≤2 5 *all samples fortified at 0.01 mg/kg, only kiwifruit fortified at multiple levels

• System suitability: o Text includes description that chlorate and perchlorate must be baseline separated. Suggest including a minimum retention time/factor as well to prevent a user from proceeding when one or both compounds are minimally retained by the column. o Table 7: relative retention time and acceptance limits provided for chlorate and perchlorate with respect to their labeled internal standards. o Table 8: expected MRM ratios and acceptance limits for chlorate and perchlorate with respect to their labeled internal standards

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1. Is the Validation Study Report in a format acceptable to AOAC? Yes, although I would like to see additional information describing the results for each sample used to determine recovery and between day precision. 2. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? Yes, although I recommend changing the “correction/conversion factor” to a “dilution factor” or “sampling factor” for improved clarity. 3. Are the figures and tables sufficiently explanatory without the need to refer to the text? Tables 9-12 should have additional information in the header/footer to describe in more detail number of samples, types of samples, etc as described in the text. 4. Are all the figures and tables pertinent? All but Table 3 (see below) 5. Could some be omitted and covered by a simple statement? Table 3 can be removed and described by a statement in the text. The method is not gradient elution as described if the composition is 50:50 mobile phase A:B for the duration of the experiment; this is isocratic elution. 6. Are the references complete and correctly annotated? Yes 7. Does the method contain adequate safety precaution reference and/or statements? Numerous statements are included to direct the user to relevant SDSs or chemical use manuals, which I feel is adequate. Recommendation: This method is highly promising but needs validation using additional matrices to meet one or more of the matrix categories listed in SMPR 2021.001 (Table 1). I recommend that the authors select one or more categories to conduct additional validation experiments and resubmit for ERP consideration.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method: AOAC Official Method Chlorate-01

Title: Chlorate and Perchlorate in Baby Foods, Infant/Adult Formulas, and Their Ingredients LC-MS/MS Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM Reviewer Name: Reviewer 3

Summary of Method: Samples are extracted using acetonitrile and 2% formic acid in water. The supernatant is subjected to weak anion exchange solid phase extraction, eluted with 5% ammonia in acetonitrile then concentrated. LC- MS/MS is used to quantitate the concentration of chlorate and perchlorate in the extract. Analytes are reported as the free anion. Isotopic dilution is used for quantitation.

Method Scope/Applicability:

The method was validated using a wide range of matrices that cover all matrices in the SMPR. See below for matrices mentioned in manuscript. The validation was performed on fortified matrices including several dairy product matrices, whey protein hydrolysate, whole milk powder, lactose, whole milk, infant formula, anhydrous milk fat (AMF), acid casein, sodium caseinate, milk protein concentrate, a number of fruit, vegetables and grains including pome fruit, musa fruit, fruiting vegetables/cucurbits, stem and stalk vegetables, root and tuber vegetables, dried legumes and pulses, high acidity fruit, stone fruit, brassica vegetables, fresh legumes, oily fruits, citrus fruits, dried fruit, grass and foliage, bulb vegetables, leafy vegetables and fresh herbs, fresh fungi, cereal grain, small fruit and berries, vegetable powders, and fruit and vegetable based baby food, meat based baby food, cereal based baby food, and other commodities.

General comments about the method:

Method is well written and easy to follow. Concerns about the method meeting the SMPR requirements for LOQ in baby food and infant formulas. Instrumentation: Instrumentation is a standard LC-MS/MS system all other instrumentation is common to analytical laboratories.

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Method Clarity: As stated previously the method is well written and easy to follow. The procedure is well defined, and the calculations are straight forward. All necessary equipment is defined. Minor confusion with the correction factor (CF) would like to hear the authors explain this with more clarity.

Pros/Strengths: • Significant amount of data collected and presented on a wide variety of matrices. • Straightforward sample preparation. • Use of isotopically labeled internal standards.

Cons/Weaknesses • Method does not appear to meet the SMPR requirements for LOQ in infant/adult formulas.

Supporting Data • General Comment: In Table 12. The perchlorate results for many Infant formula samples are reported as <0.010 mg/kg. This does not meet the SMPR requirements for LOQ.

• Performance Characteristics:

Analytical Range:

LOQ:

Accuracy/Recovery:

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Precision (RSD r ):

Reproducibility (RSD R ):

• System suitability:

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1. Is the Validation Study Report in a format acceptable to AOAC? Yes

2. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? Yes

3. Are the figures and tables sufficiently explanatory without the need to refer to the text? Yes

4. Are all the figures and tables pertinent? Yes

5. Could some be omitted and covered by a simple statement? N/A

6. Are the references complete and correctly annotated? Not Sure?

7. Does the method contain adequate safety precaution reference and/or statements? Yes

Recommendation: I would like to see LOQ data for Infant/Adult formulas. It appears that the LOQ for these samples in reported as < 0.01 mg/kg which does not meet the SMPR. It seems like the method could meet the SMPR requirements, but would need to see data that confirms this.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method: AOAC Official Method Chlorate-01

Title: Chlorate and Perchlorate in Baby Foods, Infant/Adult Formulas, and Their Ingredients LC-MS/MS Author: ADELLE K. NANCEKIVELL, ELSA M. LOTZ, LISA A. GRAHAM Reviewer Name: Reviewer 4 Summary of Method: The method involves an extraction with MeCN (2% formic acid, in water) and clean up by anion exchange SPE with 5% ammonia in acetonitrile and extract concentration. Then, analytes are separated, detected and quantified by LC-MS/MS, using isotopic dilution external calibration. Method Scope/Applicability: it is stated that LOQ is ≤ 0.007 mg/kg which is close but higher than what is required in the SMPR table 2 for most of the commodities. However, in the manuscript is stated that a linear response in the 0.005 to 1 mg/kg are achieved (abstract, scope, etc.). However, the method LOQs is stated for a reduce number of matrices and there is no information in their estimation. Validation results data are poorly presented and only for a reduce number of matrices. General comments about the method: Guard column stationary phase is from a completely different nature as the analytical column, so it may not do the expected work (safeguard the analytical column). Better use a similar stationary phase on the guard column if available, or guard columns based on inert filters with right porosity. Instrumentation: The device for concentration under N2 does not need to be necessarily capable of blowing 15 tubes at the same time. Has the inertness of the syringe filters used tested by the authors? (Micro Analytix Part Number: 17NP045AN). Preparation of Solution of Ammonia in MeCN needs to be corrected (either amounts or concentrations are not right).

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(d) Fortification. – Not clear what D.(b)(1) and D.(c) stand for.

Chromatographic peaks are very broad and with a bad peak shape indicated poor chromatography. This could be a problem as broad peak with low resolution may be interfered by matrix components. Further more sharp peaks would improve the sensitivity of the method. Chromatography is poor with very broad and bad-shaped peaks. This decreases the sensitivity of the method and increases the chances of matrix interference due to low resolution. Method Clarity: The analytical procedure is quite clear; however calculations need to be clarified. They become a bit complicated to follow with several intermediate correction factors (i.e. SR, CF, etc.) and considering volumes of intermediate extractions steps. In my opinion would be easier just to take into account that concentration of IS in the injection vials is 10 mg/L for sample extracts and 0.1 mg/L for calibration standard solutions.

What was the criteria for selecting those conversion factors (Table 6).

Other points that need some clarification are:

What is the reason to add the MeCN split in two to the sample for extraction?

Extraction, step (b) remove the word “may” by “should” or “can”. Extraction, step (c), line 4: chilling samples in

Not clear what G.(c) stand for.

Pros/Strengths: • It does not involve analytes derivatization. • Methodology is simple. • Instrumentation, materials and reagents is regularly employed in most laboratories working for the determination of contaminants.

Cons/Weaknesses • Validation study poorly described. • Weak validation data provided. • Chromatographic peaks very broad and with very bad peak shape

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Supporting Data • General Comment:

Abstract: not clear what does it mean ranges for determinations (i.e. 15-31 determinations of chlorate and perchlorate in 5-26 in different test materials; recoveries determined for 5-26 test materials…)

Method Optimization:

No information about optimization of the analytical procedure, except for the sample intake, is included on the manuscript.

• Performance Characteristics: Information about the validation plan as well as about validation results is very poor and far from meet the SMPR requirements. Nothing about some of the “minimum representative matrices” specified in the SMPR is mentioned in the manuscript i.e.: ceral-based, fruit/vegetable-based and meat-based baby food (finished products), baby food ingredients, plant based infant/adult formulas, soy protein isolate, oil/fat, lactose, maltodextrin. Although some of the matrices are covered by interlaboratory proficiency testing (Table 12), for many of them no z-score is reported, so no proficiency can be proved. No information on the validation plan is given. The manuscript should contain information on what matrices were included in the validation study, as well as the concentration levels spiked and the number of replicates for repeatability and reproducibility. LOQ concentration level should be validated (recovery and RSD within appropriate range and limits respectively). Table 10 include values of LOQ for whole milk powder, whole milk and kiwi, whereas table 11 include values on precision, recovery and uncertainty for dairy products and for a pool of fruit, vegetable, grains and baby food. A new table specifying all the matrices spiked during the validation study, and spiked concentration levels and replicates per concentration/matrix combination should be included. Validation results (recovery, repeatability and reproducibility) should be indicated per individual matrix and concentration level. LOQ should be validated to ensure good recovery and precision at this level.

Information on how the uncertainty was estimated should be included.

Analytical Range: according to Table 9. 0.05-5 mg/kg for chlorate and 0.01-1 mg/kg for perchlorate. The analytical range inferior limit should include the LOQ (linearity from LOQ should be ensured).

LOQ: only specified for Whole Milk Powder, Whole Milk and Kiwifruit. In some cases slightly out of SMPR target values.

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Accuracy/Recovery: Information for only Dairy products, and a pool of Fruit, vegetables, grains and baby food provided. Table 11 includes a column for recovery (is that the mean of recoveries??) and other for recovery range.

Precision (RSD r ): same as for recovery

Reproducibility (RSD R ): same as for recovery

• System suitability:

Based on the information provided and supporting data the method does not fulfil the SMPR.

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1. Is the Validation Study Report in a format acceptable to AOAC? No. A lot of information is missing. 2. Is the method described in sufficient detail so that it is relatively easy to understand, including equations and procedures for calculation of results (are all terms explained)? No. procedures for calculation need to be clarified. 3. Are the figures and tables sufficiently explanatory without the need to refer to the text? No. Those for validation results are incomplete and confusing. 4. Are all the figures and tables pertinent? Yes. 5. Could some be omitted and covered by a simple statement? No. 6. Are the references complete and correctly annotated? Yes. 7. Does the method contain adequate safety precaution reference and/or statements? Yes.

Recommendation: Mayor Revision : - Clarification and simplification of the calculations. - Information on method optimization should be included.

- Improvement of description on the validation study plan and validation results. - Improvement of the chromatography for both analytes would be desirable. - Improvement of the chromatography is desirable.

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