Low Lactose ERP - Review Book

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EXPERT REVIEW PANEL FOR LOW LACTOSE PRODUCTS

SHERATON DENVER DOWNTOWN 1550 Court Place, Denver, Colorado, USA CONFERENCE ROOM: Governor’s Square 12 3:00am – 7:00pm Mountain Summer Time e -Registration Opens at 2:30pm

CONTACT: CDENT@AOAC.ORG

Expert Review Panel for Low-Lactose Methods

Tuesday, September 10, 2019 3:00PM – 7:00PM MDT

Sheraton Downtown Denver 1550 Court Place, Denver, Colorado, 80202 Room: Governor’s Square 12

AGENDA

1. Welcome and Introductions Sean Austin, Nestlé Research Centre, Chair of the Low-Lactose Expert Review Panel

2. Review of AOAC Volunteer Policies & ERP Process Overview and Guidelines Deborah McKenzie, AOAC INTERNATIONAL

3. Review of Methods For each method, the assigned ERP members will present a review of the methods and manuscripts, after which the ERP will discuss the method and reach consensus on the status for each method. A. Candidate Method LAC-001: Lactose, free and low, Profile by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD): Single- Laboratory Validation (SLV) Investigation a. Primary Review: John Szpylka, Mérieux NutriSciences b. Secondary Review: Sookwang Lee, US FDA c. Tertiary Review: Mehmet Gumustas, Ankara University Institute of Forensic Sciences d. Discussion and Consensus B. Candidate Method LAC-002: Quantitation of Six Common Food Sugars in Various Matrices by HPLC-MS a. Primary Review: Salvatore Parisi, Al-Balqa Applied University

Draft meeting agenda subject to change without notice

AOAC Low Lactose Expert Review Panel Draft Agenda

b. Secondary Review: Marina Torres Rodriguez, LATU c. Tertiary Review: Tomasz Tuzimski, Medical University of Lublin d. Discussion and Consensus

C. Candidate Method LAC-003 : LAC-003: BIOMILK 300 LAC a. Primary Review: Jinchuan Yang, Waters

b. Secondary Review: Michael Goodrich, Cornerstone Analytical Labs c. Tertiary Review: Sudhakar Yadlapalli, First Source Laboratory Solutions d. Discussion and Consensus

D. Candidate Method LAC-004: LactoSense®R

a. Primary Review: Martine van Gool, FrieslandCampina b. Secondary Review: Lucien Duchateau, DSM Food Specialties c. Discussion and Consensus

E. LAC-005: Determination of Sugars in Animal Feed, Pet Food, and Human Food applying Ion Chromatography with Pulsed Amperometric Detection (IC-PAD) a. Primary Review: George Joseph, AsureQuality New Zealand

b. Secondary Review: Angelika Semmler, Chr Hansen c. Tertiary Review: Sean Austin, Nestlé Research Centre d. Discussion and Consensus

F. LAC-006: K-LOLAC Enzymatic Low Lactose Assay a. Primary Review: David Ellingson, Eurofins Food Integrity and Innovation b. Secondary Review: Brian Lang, US NIST c. Tertiary Review: Nour Eddine Es-Safi, Mohammed V University in Rabat d. Discussion and Consensus

4. Final Action Requirements for Adopted Methods (if applicable)

5. Adjourn

LOW LACTOSE METHODS EXPERT REVIEW PANEL

METHODS AND SMPR ACCESS

• AOAC SMPR 2018.009 • METHODACCESS [ERP ONLY - PASSWORD REQUIRED]

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-05 18:12:35

Name

John Szpylka

E-mail

john.szpylka@mxns.com

Organization

Merieux NutriSciences

Title of Method

Lactose, free and low, Profile by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD): A Single- Laboratory (SLV) Investigation

AOAC Candidate Method Number (e.g. ALN-01)

LAC-001

Applicable SMPR

2018.001

I. Summary of the Method

The method is based on a broader multiple sugar analysis with this method submission focusing on the lactose disaccharide. For each sample, two subsamples are used for this analysis. The method abstract states sugars are extracted from both solutions using an aqueous-ethanol solution; however the method protocol only states water is used. The ethanol must be confirmed since ethanol is needed to denature enzymes present in the sample, a criteria listed in the SMPR. After extraction, the solutions are clarified using Carrez solutions. One subsample is treated with B-galactosidase to remove lactose. After HPAEC-PAD separation/quantitation, the difference in the peak areas at the lactose retention time is used to quantify the lactose in the sample. All SMPR criteria are met; however, the method protocol only states water is used to extract lactose. Since possible conflicting enzymes could decrease lactose recovery, the use of ethanol as listed in the abstract must be confirmed. The authors did account for an error in the SMPR LOD (stated as 5 mg/100mg or 5%) with the method achieving an LOD of 0.005% lactose.

The technique has the required sensitivity and accounts for potential lactose chromatographic co-elutants.

3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used.

RSDR was not correctly defined in the validation but the data still met the SMPR requirement.

RSDR data was collected as per decade duplicate analyses of 9 different samples on 9 different days with individual duplicate values measured on different days. The protocol lists these analyses are done at the same location on the same instruments/equipment but with two analysts. I read this as intermediate precision. However, RSDR is typically attained in the process after First Action Status is granted, so the resulting MLV can be used for this SMPR requirement.

As a note: data for RSDr (repeatability) was also collected in a second approach of deco-duplicate analyses of 9 samples on the same day (correct).

Both approaches estimated RSDr as 9.3% and 7.9%, both below the RSDr requirement of 10%

The remaining definitions were met. Notably reported lactose levels account for monohydrate forms.

This condition is met. I especially approve of the Safety Table 5 which presents safety concerns in a clear an actionable fashion.

The method abstract states sugars are extracted from both solutions using an aqueous-ethanol solution; however the method protocol only states water is used. The ethanol must be confirmed since ethanol is needed to denature enzymes present in the sample, a criteria listed in the SMPR. This could impact the method.

RSDr issues are described above but data still meets SMPR requirements.

Authors stated the SMPR CRM of SRM 2383a was not available. An SRM substitute, the SRM1869a from the SPIFAN II SLV kit was used after approval of the lactose content given in the reference certificate for NIST SRM1869 with that measured with the present method. Also used were two MUVA Kempten CRMs (UHT milk at 0.006% lactose and sodium casseinate (0.0024% lactose). All results were acceptable.

3. Is there information demonstrating that the

The method meets the lactose specified SMPR requirements.

method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

No additional steps recommended.

Recommended peak resolutions for potential conflicting compounds are listed to assess chromatographic separations. This is also listed as a tool to adjust chromatographic conditions to attain proper peak separations.

The method validation included an in-depth robustness study to assess method performance. The controls and suitability parameters in the method are acceptable.

1) The extraction protocol must be corrected for the extraction solution being used (water or water-ethanol). 2) One step is listed as adding lactose to one of the 2 subsamples. If this is to assess enzyme performance, it should be stated in the method along with acceptance criteria. 3) There may be some Eurofins' nomenclature in the submitted method (such as PD5954 Procedure for the handling of control charts). These should be removed. 4) The validation raw data was not easy to decipher given the verbiage. This should be clarified especially for the LOD determination. Method can measure low levels of lactose with an LOD that can be used to declare a food as lactose free. Potential co-eluting interferences are negated using the the difefrence between 2 subsamples with one subsample enzymatically removing lactose prior to measurement.

5. Based on the supporting information, what are the pros/strengths of the method?

6. Based on the supporting information, what are the cons/weaknesses of the method?

Method uses equipment that can be fussy at time, but the method's quality checks will monitor its performance.

7. Any general comments about the method?

Points that need addressing: 1) The method abstract states sugars are extracted from both solutions using an aqueous-ethanol solution; however the method protocol only states water is used. The ethanol must be confirmed since ethanol is needed to denature enzymes present in the sample, a criteria listed in the SMPR. 2) Adjust the SLV report to properly report RSDr and RSDR as stated in the above section. 3) The validation raw data was not easy to decipher given the verbiage. This should be clarified especially for the LOD determination. 4) One step is adding lactose to one of the 2 subsamples. If this is to assess enzyme performance, it should be stated in the method along with acceptance criteria.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

I recommend the method for adoption as First Action if the general comments described above are satisfactorily met.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-09 09:05:20

Name

Sookwang Lee

E-mail

Sookwang.lee@fda.hhs.gov

Organization

US FDA

Title of Method

Lactose, free and low, Profile by High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD)

AOAC Candidate Method Number (e.g. ALN-01)

LAC-001

Applicable SMPR

SMPR 2018.009

I. Summary of the Method

SLV study of a HPAEC-PAD method for quantification of lactose, based upon IDF/WD244 and ISO /CD 22184. All the criteria within the SMPR2018.009 are appropriately addressed. The report excellently describes the lactose analysis in low-lactose and lactose- free milk products, which is challenging, partly because HPLC-RID is not sensitive enough, and the poor chromatogram baseline by IC in some samples (milk), among others. The reviewer couldn’t find the representative chromatograms for the real milk samples in the report. How was the baseline? This method chose the best option (No 3) for the lactose analysis in free-, or low- lactose milk products. 1) direct measurement (HPLC-RID?), 2) Indirect (?) measurement via reduction to the sugar alcohols, first, which will not be a problem in the most dairy products, but would be problematic in low lactose milk products and 3) Direct and indirect measurements via D-glucose (?) and comparing the differences.

Only drawback of this method is that there will not be many labs capable of running IC methods routinely.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing.

Yes, especially this method sufficiently demonstrates the key information regarding how to handle/address potential adulterants, listed in Table 1 of SMPR 2018.009, at the very least. The reviewer considers that the method’s capability to demonstrate adequate separation of potential interferents as essential. Two groups of compounds (sugar alcohols and lactose analogs) amongst those listed in the Table 1 of SMPR 2018.009 are important. The report provided the detailed explanation under the section of Selectivity and interferences (pp. 28 ~ 31). A. This method stated that the most sugar alcohols eluted earlier than the analyte. Table with the retention times is provided, but no chromatogram is provided in the report. B. Utilization of more specific β -galactosidase could be considered. How do you discern the contribution from lactose analogs vs. that of lactose? The reviewer is not 100% certain whether β -galactosidase used in the method would not cleave lactulose and/or 1,6- β -D-allolactose (few examples of lactose analogs among others) to results in D-glucose and/or D-galactose? β -galactosidase from E. coli (Section B. 8 of page 8 on the report) is reported as non-specific or less specific in the literature. D-galactosyl-D-glucose disaccharides (or oligosaccharides) might form during the production of low-lactose or lactose-free milk products (transglycosylation). C. Table 10 of page 15 lists chromatographic resolution between lactulose vs. lactose.

2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s).

Yes

Mostly, yet somewhat rudimentary (and redundant) information regarding how to handle the eluents and wastes during the IC analysis could be added.

III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 3. Is there information demonstrating that the

Yes. LOD, Accuracy, Recovery, Repeatability, among others are well defined and explained properly.

Yes.

Per Table 2 of SMPR, 3 different ranges of lactose contents are specified. The results from LAC-001 demonstrates that the method performances were satisfactory in all 3 analytical ranges, 4 samples for the lower range of lactose content, 5 samples for the mid-range (10 ~ 100 mg/100g) and 4 samples for the higher range. The focus of this method should be on low level of lactose, i.e., around 10 mg/100g. The report shows that the method LAC-001 performs well in that range of lactose content. Table 3 in SMPR states the NIST SRM2383a (baby food composite), which is not found in LAC-001. Two CRMs of MUVA-ML-2311 and MUVA-CA-0904 were used. The results (Table 17) match well with Reference values of lactose monohydrates. The results for galactose and glucose (0.24 mg/g, respectively) from MUVA-ML-2311 could have been reported as well (although it’s not under the scope of the method).

Yes.

Not really.

2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions. 5. Based on the supporting information, what are the pros/strengths of the method? 6. Based on the supporting information, what are the cons/weaknesses of the method?

Yes. System suitability is presented. Method uncertainty is well defined and calculated, with the reported extended uncertainty at 18.9%. The results from the Method LAC-001 were compared with an alternative method. The reviewer assumes that the identical sample extraction procedure was adopted, and the sample extracts were analyzed by a LC-MS/MS method.

Yes. Addressed in the previous Question 2.

Although some of the sentences could be written better (or English translation needs to be more specific), the report is well written.

Pros: This method seems to be capable of delineating almost all the potential interferants listed in SMPR.

Cons: Only drawback of this rather a laborious method is that there will not be many labs capable of running IC methods routinely, esp. in the developing countries.

7. Any general comments about the method?

N/A

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

The reviewer strongly recommends this method be adopted as a First Action, based upon the rationale that this method demonstrated its superior (?) sensitivity of lactose in free- or low-lactose milk products and the selectivity (against potential interferants).

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-09-04 04:58:22

Name

Salvatore Parisi

E-mail

drparisi@inwind.it

Organization

Al Balqa Applied University

Title of Method

Quantitation of Six Common Food Sugars in Various Matrices by HPLC-MS

AOAC Candidate Method Number (e.g. ALN-01)

LAC-002

Applicable SMPR

Low Lactose AOAC SMPR 2018.009

I. Summary of the Method

This method has been proposed with the aim of determining six common mono and disaccharides (fructose, galactose, glucose, sucrose, maltose, and lactose) in various food, infant formula, feed and pet food matrices. The method can be evaluated with relation to AOAC SMPR 2018.009: Standard Method Performance Requirements (SMPRs®) for Lactose in Low-Lactose or Lactose Free Milk, Milk Products, and Products Containing Dairy Ingredients. It has to be noted that this approach concerns six carbohydrates including lactose. Consequently, another SMPR - AOAC SMPR® 2018.001 (Standard Method Performance Requirements (SMPRs®) for Sugars in Animal Feed, Pet Food, and Human Food) is applicable. In fact, Authors note that the method is also a re-submission for SUG-004. Basically, samples are extracted in 25% aqueous reagent alcohol at 60 °C under sonication and shaking. Carrez reagents are then introduced to precipitate protein. After centrifugation, the supernatant is diluted accordingly before LC-MS analysis. The method limit of quantitation (LOQ) is 0.005% (50 ppm) for each individual analyte. Yes, the applicability of the method supports the applicability of the SMPR. I would suggest the inclusion of “Low-Lactose or Lactose-Free” products in the “Purpose” field when speaking of “various food, infant formula, feed and pet food matrices”, with reference to AOAC SMPR 2018.009, section 2 (Applicability).

Yes, the analytical technique used in the method meets the SMPR.

In general, the definitions specified in the SMPR are used and applied appropriately in the method. On the other hand, Limit of detection (LoD) and Repeatability (expressed as repeatability standard deviation or SDr) are not mentioned. Reference: AOAC SMPR 2018.009, section 4 (Definitions).

4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s). III. Review of Supporting Information 1. Are the definitions specified in the SMPR used and applied appropriately in the supporting documentation (manuscripts, method studies, etc...)? If not, please explain the differences and if the method is impacted by the difference. 2. Is there information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Materials stated in the SMPR? If not, then specify what is missing and how this impacts demonstration of performance of the method. method performs within the SMPR Method Performance REquirements table specifications for all analytes in the SMPR applicability statement? If not, please specify what is missing and whether or not the method's applicability should be modified. 3. Is there information demonstrating that the

In general, the method contains appropriate precautions and warnings. I would suggest to mention AOAC Official Methods of Analysis (2005), Appendix B: Laboratory Safety.

Yes, the definitions specified in the SMPR are used and applied appropriately in the supporting documentation.

Reference materials stated in the SMPR are: MUVA ML-2308 (UHT-Milk, low in lactose) and ML-2309 (UHT-Milk, lactose-free, frozen), and MUVA CA-0904. The candidate approach mentions correctly available MUVA Kempten materials: MUVA ML-2310 and ML-2011, and ML-2309.

Yes, there are specific information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications. Actually, the method concerns six sugars, while AOAC SMPR 2018.009 concerns only lactose. In addition, it has to be noted that:

a) Reproducibility tests have been performed for lactose with relation to the following samples: Cheese Spread only (repeatability is not mentioned)

b) Precision tests have been performed for lactose with relation to the following samples: dry pet food only

c) A specific extraction robustness study has been performed for lactose with reference to : lactose-free vanilla yogurt

d) Finally, the following reference standards have been considered for lactose and with reference to AOAC SMPR 2018.009: MUVA ML-2310 and ML-2311, and ML-2309.

IV. General Submission Package 1. Based on the supporting information, were there any additional steps in the evaluation of the method that indicated the need for any additional precautionary statements in the method? 2. Does the method contain system suitability tests or controls as specified by the SMPR? If not, please indicate if there is a need for such tests or controls and which ones. 3. Is there information demonstrating that the method system suitability tests and controls as specified in the SMPR worked appropriately and as expected? If no, please specify. 4. Based on the supporting information, is the method written clearly and concisely? If no, please specify the needed revisions.

No.

The method includes one reference standard: MUVA ML-2311. On these bases, the LOQ is calculated. Actually, it appears that specific blanks have not been included. Also, the LOD should be calculated (in the text, LOQ is calculated on the basis of MUVA ML-2011 results; LOD should be mentioned because it is three times standard deviation).

The method system suitability tests and controls (as specified in the SMPR) are fit for purposes. However, it appears that specific blanks have not been included.

Yes, the method is written clearly and concisely. I would only suggest three revisions:

a) The method is considered for lactose, but six sugars are mentioned (and lactose is not analytically considered in all samples). Probably, more emphasis should be shown with reference to lactose only, and with concern to “milk, milk products, and products containing dairy ingredients that are low-lactose or lactose-free” (reference: AOAC SMPR 2018.009). Samples might be variegated

b) The above-mentioned SMPR requires that repeatability is demonstrated; in addition, blanks should be specifically mentioned for controls

c) There are specific references to particular equipment in the text. Could these references be clearly expressed with alternative choices as follows ? “HPLC column, per example ……) OR EQUIVALENT ... " The same thing could be reviewed when speaking of the final summary with relation to column performances: “the …….. column AND EQUIVALENT COLUMNS provide excellent separation…”

In this way, references to specific materials/equipment and equivalent systems would be more evident, even if the section: Equipment states it.

5. Based on the supporting information, what are the pros/strengths of the method?

Pros/strenghts of the method are surely rapidity and reliability. Also, the method appears user-friendly and easily readable.

6. Based on the supporting information, what are the cons/weaknesses of the method?

In my opinion, the emphasis should be enhanced with relation to lactose and “milk, milk products, and products containing dairy ingredients that are low- lactose or lactose-free” (reference: AOAC SMPR 2018.009).

Also, repeatability should be demonstrated, blanks should be specifically mentioned for controls (reference: AOAC SMPR 2018.009).

7. Any general comments about the method?

The method clearly concerns six sugars and different products.

Should this method match requirements (reference: AOAC SMPR 2018.009), some review would be needed (cons/weaknesses of the method).

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

In my opinion, the method should be reviewed.

It is a valuable method, and some little modifications would be carried out (specific reference to lactose, low-lactose or lactose-free products; repeatability and related RSD; needed blanks; possibly, mention of AOAC OMA, Appendix B: Laboratory Safety; and –possibly – mention of needed equipment specifying clearly equivalent choices in the text).

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-08-26 19:12:47

Name

Marina Torres

E-mail

mtorres@latu.org.uy

Organization

Latu

Title of Method

Quantitation of Six Common Food Sugars in Various Matrices by HPLC-MS

AOAC Candidate Method Number (e.g. ALN-01)

LAC-002

Applicable SMPR

SMPR 2018.009

I. Summary of the Method

This method quantifies lactose, and five other sugars (fructose, galactose, glucose, sucrose and maltose) in various food, infant formula, feed and pet food matrices. Samples are extracted in 25% aqueous reagent alcohol at 60 °C under sonication and shaking. Carrez reagents are used to precipitate protein. After centrifugation, the dilutions are analyzed by LC-MS.

II. Review of the Method Only 1. Does the applicability of the method support the applicability of the SMPR? If not, please explain what is missing. 2. Does the analytical technique(s) used in the method meet the SMPR? If not, please specify how it differs from what is stated in the SMPR. 3. Are the definitions specified in the SMPR used and applied appropriately in the method? If no, please indicate how the terms are used. 4. Does the method, as written, contain all appropriate precautions and warnings related to the method's reagents, components, instrumentation, or method steps that may be hazardous? If no, please suggest wording or option(s).

Yes, the method could be used for matrices and levels mentioned in the SMPR.

The analytical technique of the method meets the SMPR.

Yes

The Reference Materials stated in the SPMR were most of them used, but:

-NIST 18492a Infant Formula: lactose level outside the scope of the SMPR. -NIST 1869 Infant Formula: the same level of lactose of NIST 2383a stated in the SMPR. Good agreement with the reference value but with only 4 replicates, the intermediate reproducibility (not calculated by the authors, the value was calculated by the reviewer) was higher than the stated in the SMPR. -MUVA CA-0904 Sodium Caseinate: only one value of this material. The recovery does not meet the SMPR. -MUVA 2310 UHT Low lactose milk: Good agreement with the reference value but with only 4 replicates, the intermediate reproducibility (not calculated by the authors, the value was calculated by the reviewer) was a little bit higher than the stated in the SMPR. -MUVA 2311 UHT Lactose free milk: Good data, 8 replicates were analyzed, good agreement with the reference value and the intermediate reproducibility meets the SMPR. There is not repeatability data. Regarding intermediate reproducibility data, it was calculated for a higher level than the stated in the SMPR (cheese spread 6,8% of lactose). The reviewer calculated some intermediate reproducibility data from the Reference Materials analyzed but data do not meet SMPR except for UHT Lactose Free Milk. Regarding recovery, it was only calculated for dry pet food sample. The recovery was calculated at 3 different levels but only one, the lowest level, must be considered. The other two are higher than the levels specified in the SMPR. The recovery for the level 1% meets the SMPR. Good agreement with some reference values of reference materials. Regarding interferences, not problems with other sugars (a lot of sugars tested) except from cellobiose. But it seems not to be an important interference for the matrices of the scope. Some authors mention that 25% ethanol is not enough for avoid enzyme activity, but the authors analyzed interference with beta galactosidase and the results were very good. Only sugars and enzymatic activity were tested, there are some potential interferences mentioned in the SMPR that were not considered. More data is needed.

3. Is there information demonstrating that the

No SST are mentioned. It could be good to add a check standard or a check sample.

No SST are mentioned.

The method is clear and well written. The voltage of the MS-capillary must be in KILO-volt?

Easy to perform. Very good resolution of lactose from other possible present sugars in the sample.

MS detectors are not present in all labs. Very expensive internal standards (labelled sugars) for routine analysis.

7. Any general comments about the method?

Good but expensive method. More data is needed.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

I do not recommend moving forward the method currently. I consider is a good method, but more data is needed.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-08-31 09:08:23

Name

TOMASZ TUZIMSKI

E-mail

tomasz.tuzimski@umlub.pl

Organization

Medical University of Lublin, Poland

Title of Method

Quantitation of Six Common Food Sugars in Various Matrices by HPLC-MS

AOAC Candidate Method Number (e.g. ALN-01)

LAC-002

Applicable SMPR

AOAC SMPR 2018.009

I. Summary of the Method

The proposed method (LAC-002) entitled ‘Quantitation of Six Common Food Sugars in Various Matrices by HPLC-MS’ described Kai Liu, Ben Pointer is applicable for the detection and quantitation of the content of six common mono and disaccharides (fructose, galactose, glucose, sucrose, maltose, and lactose) in various food, infant formula, feed and pet food matrices. The developed method was evaluated following the definitions of Standard Method Performance Requirements (SMPRs®) for Lactose in Low-Lactose or Lactose-Free Milk, Milk Products, and Products Containing Dairy Ingredients (AOAC SMPR 2018.009) with respect to Limit of Detection (LOD) and the Limit of Quantitation (LOQ), Linearity, Repeatability, Reproducibility, Recovery.

Generally: YES: The applicability of the method is adequate to the applicability of the SMPR.

YES: The analytical techniques in the method are adequate and meet the SMPR.

YES: Definitions, which are specified in the SMPR, were listed in the description, also were applied appropriately in the method.

Yes: The method contains all appropriate precautions and warnings related to the method’s reagents, components, instrumentation, or method steps that may be hazardous.

YES: The definitions specified in the SMPR were used and applied appropriately in the main document and supporting documentation (appendix A, and B, etc.).

YES: There are information demonstrating that the method meets the SMPR Method Performance Requirements using the Reference Material stated in the SMPR.

YES: There are information demonstrating that the method performs within the SMPR Method Performance Requirements table specifications for all analytes in the SPMR applicability statement.

In my opinion there is no need.

YES: There are.

YES: There are information demonstrating that the method system suitability tests and control as specified in the SMPR worked appropriately and expected.

Generally: The method is well described and substantively prepared. The project of the method is well integrated and includes a clear and concise description.

The developed method was evaluated following the definitions of AOAC SMPR 2018.009 with respect to values of LOD and LOQ, Linearity, Repeatability, Reproducibility, Recovery. Analytical data was collected for all compounds, and required / suitable matrices (such as food, infant formula, feed and pet food matrices) listed in AOAC SMPR 2018.009. In the APPENDIX A, Authors presented example chromatograms and potential interfering compounds. In the APPENDIX B, Authors wrote: The theoretical LOQ is 0.005%. MUVA ML-2311 Lactose-free UHT Milk has ~0.007% of lactose. Based on six replicate determinations of MUVA ML-2311 in a single batch, the standard deviation for lactose result was determined, and considered as the “noise level” in a true sample matrix. Therefore, LOQ can be calculated as ten times the noise.' ONLY: My additional recommends: Could Authors be evaluate more precisely matrix effect and influence of them on the results? Examples of chromatograms for some matrices have been omitted, or their suitable full results are not presented in the described method.

6. Based on the supporting information, what are the cons/weaknesses of the method?

7. Any general comments about the method?

My additional remarks and recommendations to the method are as follows:

Page 12, Extraction Robustness Study – Special Spike Recovery Results, table: In my opinion Authors may add RSD values for lactose and maltose at three spiked levels (0.1%, 2%, 20%).

Page 12, Summary In summary, the XBridge BEH Amide column provides excellent separation of six common sugars that are found in food, infant formula, pet food, and feed matrices.' In my opinion Authors may add Rs values for all analytes.

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

In my opinion, the AOAC Candidate Method LAC-002 entitled ‘Quantitation of Six Common Food Sugars in Various Matrices by HPLC-MS’ described Kai Liu and Ben Pointer is applicable for the detection and quantitation of the content of six common mono and disaccharides (fructose, galactose, glucose, sucrose, maltose, and lactose) in various food, infant formula, feed and pet food matrices. The AOAC Candidate Method LAC-002 can be recommend for adoption as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL.

AOAC SMPR ERPs - 2019 METHOD REVIEW FORM

Submission Date

2019-08-19 10:24:52

Name

Jinchuan Yang Yang

E-mail

jinchuan_yang@waters.com

Organization

Waters

Title of Method

BIOMILK 300 LAC

AOAC Candidate Method Number (e.g. ALN-01)

LAC-003

Applicable SMPR

2018.009

I. Summary of the Method

This method is a proprietary method for determining the lactose in low lactose or lactose-free products. The measurement of lactose involves an enzymatic electrochemical biosensor (BIOMILK 300 LAC). This biosensor incorporates a biochemical sensing element and a transducer system that can relates the concentration of lactose to a measurable signal. The analysis is performed directly over the aqueous solution. The addition of the sample (or the lactose) in the solution causes sequential redox reactions that involve a release of electrons proportional to the concentration of the lactose. Such the concentration of the lactose is determined. The applicability of the method meets the SMPR requirement. Sample type include milk, yogurt, cream, soft cheese, infant formula, Café latte, and protein shake.

There is no requirement of any specific technique in the SMPR. So, it is a yes.

Yes

Yes, the method description contains a Safety Precaution section, in which the potential safety hazards are highlighted.

Yes. (Please note that the equation it used to estimate the LOD is not common).

The SMPR specifies a reference material, NIST SRM 2383a, and Harmonization materials from National Milk Laboratories (UK) and from MUVA (Germany). The supporting data of this method shows results from four MUVA samples, the MUVA-ML-2308, 2309 (best used before: 06/2018, and the MUVA-ML-2310, 2311. The intermediate reproducibility from the determination of the four harmonization samples meet the SMPR requirement. However, the recovery was not evaluated. The reason given in the supporting document says that “the reference values of the materials were obtained from two different technologies, enzymatic assays, and HPLC, and none of them are reference methods for low lactose products”. I did a simple calculation to compare the HPLC values from the harmonization samples to the BIOMILK lactose results. We can see that for the high concentration (>100 mg/100g) lactose samples (-2308 and -2310 in Table 17), the recovery is within 10%, meet the SMPR. But for the lactose-free samples (- 2309 and -2311), the BIOMILK results are outside the SMPR limit on recovery. However, please note that even though the BIOMILK results are not within the SMPR limit on recovery for lactose-free samples, they are within the HPLC reference values’ uncertainty limits. In the supporting document, it contains a third-party validation report, in which the recovery was evaluated against a MUVA-ML-2311 UHT milk, which has a reference value of lactose 0.0066% (m/m), a recovery of about 68% was obtained. In that report, a conclusion was made that this level of bias was not significantly different from the reference value. However, this recovery is outside of the SMPR.

3. Is there information demonstrating that the

Repeatability on different matrices are within 10%. The samples include UHT milk, sugary plain yogurt, flavoured plain yogurt, Greek yogurt, flavoured liquid yogurt, chocolate milk, cream, soft cheese, infant formula, café late, high protein vanilla shake. Recovery on lactose was evaluated in spiking experiment. The results are good. LOD/LOQ results are good. ML harmonization materials were measured, but recovery was not calculated. Recovery in each concentration range, especially the low lactose range 5-100 mg/100g, should be evaluated with reference materials.

None

The SMPR requires blanks and check standards. The method specifies that every 10-15 measurements or after the device has not been running for a minimum of 30 min, the operator need to do a Standard measurement, which serves as a check standard.

It is a proprietary method. All the calibration and check are done internally. There is no raw data available to assess whether the system suitability tests are adequate or not.

The description of the method is clear. Note: the g.3 section is not clearly marked.

The strengths of this method include: No sample extraction and filtration needed as compare to other chromatographic method; No mobile phases, no data process, the concentration result is directly given by the device after the measurement.

6. Based on the supporting information, what are the cons/weaknesses of the method?

The potential weakness of the method include: 1) It needs a prior knowledge of the unknown sample matrix, such as the glucose level in the sample, and whether ascorbic acid exist in sample or not, to determine how the sample is treated, and how the device is calibrated. 2) For low lactose concentration range (5-100 mg/100g), the recovery result seems not within the SMPR limit of 85-115%. 3) It is a proprietary method, do we know if any alternative source of the device and the chemical solutions are available or not? This method uses a simple device to measure the lactose level. It is very sensitive, can reach to the required LOQ. Also, there is no chromatography needed, so less sample prep procedure, and it is easy to use. Anyone who can use a pH meter should be able to operate this device. However, there are lots of chemicals or interferants that can contribute to the device signal. Some chemicals have very low response factors, but some have very high response factors, such as ascorbic acid, and vitamin Bs. The method tries to remove the background signal by a special sample treatment for ascorbic acid, and by changing how the calibration standard and Measurement Standard are prepared. This approach might be OK for samples with relatively high lactose range, but not adequate for low lactose range. Because it is not possible to accurately estimate how much signal is from the unknown sample matrix, and how much is from the lactose. I think this method is a very sensitive, reliable, and easy to use method to determine lactose concentration in milk and milk products for medium to high lactose concentration range. I am not confident that this method can accurately determine the lactose concentration in lactose-free products. This method also needs to know the glucose level in samples, and whether the ascorbic acid is in presence or not, which limited its use. The third concern is that it is a proprietary method, we need to find out if any alternative device is available, and if there is alternative way to prepare the solutions? I can not recommend this as a First Action method.

7. Any general comments about the method?

V. Final Recommendation Do you recommend this method be adopted as a First Action and published in the Official Methods of Analysis of AOAC INTERNATIONAL? Please specify rationale.

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