AOAC SPIFAN Nutrients ERP Reviewer Forms (Amino-05)

AOAC Stakeholder Program on Infant Formula and Adult Nutritionals

NUTRIENTS REVIEWER FORMS (AMINO-05)

Thursday, July 28, 2022

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_ Amino 05

Title: An Ion Exchange Liquid Chromatography with Post Column Derivatization Method for the Determination of Taurine in Infant Formula and Adult/Pediatric Nutritional Formula

Author: Mérieux Reviewer Name:

Reviewer 1 Summary of Method: Powdered, semi liquid or liquid samples are extracted with a citrate buffer in the presence of Norleucine as internal standard. The extracted reacted samples are filtered and analyzed using Amino Acid Analyzer ionic chromatography with post column derivatization with Ninhydrin.

Method Scope/Applicability: Determination of Taurine amino acid in infant formula and adult/pediatric nutritional formulas

General comments about the method: This method uses well-established methodology for analyzing amino acids using cation-exchange column and post-column derivatization with Ninhydrin reagent. The authors also submitted a method that can be used to analyze free and total amino acids but the validation study was completed for Taurine only. For Taurine a simple extraction sample preparation procedure was used but hydrolysis step is required to determine the total amino acids. The chromatographic conditions described in the method are unique to a particular amino acids analyzer so buffers and Ninhydrin reagent have to be purchased from the manufacturer of the analyzer. Different brands of amino acids analyzers are available on the market and their performance should be comparable to the one described in the method. The method should emphasize general performance requirements that can be used to assess the performance of different amino acids analyzers.

Method Clarity:

Method is clearly written

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Pros/Strengths: Well-known analytical approach using Ninhydrin reagent for analysis of amino acid. The same chromatographic conditions can be used to determine all the amino acids listed in SMPR but sample hydrolysis step is needed if analysis of total amino acids is required

Cons/Weaknesses The exact composition of buffers and reagents is not available so all the solutions have to be purchased from the manufacturer of the amino acids analyzer.

Long run time. If no other amino acids besides Taurine and Norleucine are present on the chromatogram it would be beneficial to optimize the method for shorter run time.

Supporting Data • General Comment:

Method Optimization:

• Performance Characteristics:

Analytical Range : 0.5 – 2500 mg/100 g for liquids or reconstituted powders – meets SMPR

LOQ: 0.5 mg/100g for reconstituted powder samples – meets SMPR

Accuracy/Recovery: meets SMPR – 2 samples with 3 spike levels were used for accuracy study

Precision (RSD r ): meets SMPR – 5 samples used for precision study

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Reproducibility (RSD R ): NA

• System suitability: Acceptance criteria for calibration curve and chromatographic separation are provided

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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?

No

6. Are the references complete and correctly annotated?

Yes

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

Recommendation: The method describing analysis of Taurine has met SMPR requirements and I would recommend it as AOAC first action official method. Notes should be made in the method that equivalent equipment, columns, buffers and Ninhydrin reagent from other manufacturers can be used, providing system suitability requirements are met. Since the validation study was only competed for Taurine the method describing analysis of free and/or total amino acids should not be considered at this time. It would be beneficial if authors could complete validation for free and total amino acids so the same method could be used for all the amino acids listed in SMPR.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method Amino-05

Title: An Ion Exchange Liquid Chromatography with Post Column Derivatization Method for the Determination of Free and/or Total Amino Acids and Taurine in Infant Formula and Adult/Pediatric Nutritional Formula Author: Giampaolo Perinello, Paolo Tonini and Lorenzo Papa. Mérieux Nutrisciences Italia. Reviewer Name: Reviewer 2 Summary of Method: The method allows the determination of free, total and sulfur amino acids and Taurine in infant formula and adult/pediatric nutritional formula. For free amino acids and Taurine samples are extracted with a citrate buffer in the presence of Norleucine as an internal standard. For the determination of total non-sulfur amino acids the samples is hydrolyzed in oven with hydrochloric acid and dilute for chromatographic analysis. For the determination of sulfur amino acids such as Cystine + Cysteine and methionine, the sample is reacted with an oxidizing mixture of formic acid and phenol to convert them into Cysteic acid and Methionine sulfone. This is followed by hydrolysis in oven with hydrochloric acid and diluted for chromatographic analysis. The extracted samples are filtered and analyzed using Amino Acid Analyzer Ionic Chromatography with post column derivation with Ninhydrin and detected with UV-VIS detector. General comments about the method: The determination is performed by extracting a portion of the sample using citrate buffer and Norleucine as internal standard. The extracted sample is analyzed using Amino Acid Analyser Biochrom 30+ with post column derivatization with Ninhydrin and UV-VIS detector at 570 nm wavelength. This procedure is applicable to powdered samples, semi-liquid or liquid samples. Although the method have a very good resolution. I consider it is inconvenient to use equipment from an exclusive supplier. Method Clarity: The method is clear, simple and easy to follow. Method Scope/Applicability: The method is performed to determine only Taurine in SPIFAN samples kit.

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Pros/Strengths: This Step Gradient system, allows to obtain the maximum reproducibility of the retention times and resolution of the analysis and allows one hour saving in analysis times compared to other standard methods for amino acids. The ion exchange analytical column is completely regenerable, so it does not have to be periodically replaced and allows a workflow exceptionally low (25ml / hour) for a reduced consumption of eluent and reaction solvents that means a significant reduction of the analysis costs.

Cons/Weaknesses

Equipment from exclusive supplier.

Supporting Data • General Comment:

Method Optimization:

Three calibration curves have to be tested with independently prepared standards. For testing the calibration models 6 calibration samples are used ranging from 0,1 – 11 mg/L. Working samples and calibration samples are both analized in the Amino Acid Analyser Biocrom 30+ with post column derivation with Ninhydrin.

• Performance Characteristics:

Analytical Range: SMPR: 0.5–2500 mg/100 g reconstituted final product it is necessary to describe the calculation procedure.

LOQ: SMPR: ≤0.5 mg/100 g reconstituted final product LOQ : 0.43 mg/100g for reconstituted powder samples.

The laboratory has proceeded by preparing solutions at least at four concentration levels between LOD and two times LOQ. Three different runs using different instruments and from three different analyst were conductes in order to evaluate LOQ.

Accuracy/Recovery : SMPR: 0.5–5.0

±12% 5.0–150 ±10% 150–2500 ±7%

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The NIST SRM 1869 was analized through spikes at two ranges in the middle/high field of SMPR. Spiked and unspiked samples were analyzed in triplicate on each three days. Results: 99.6 % ±12% for low Taurine content (0.5-5.0)

96.8 % ±3.2% for medium Taurine content (5.0-150) 98.4 % ±3.1% for high Taurine content (150-2500)

Precision (RSD r ): SMPR: 0.5–5.0

≤7%

5.0–150 ≤5% 150–2500 ≤3%

The precision was assessed by repeated measurements carried out on 6 different days, in duplicate on the each sample of SPIFAN kit. The precision was evaluated also on spiked samples NIST 1869 and NIST 8261 to cover all the analytical range. Results:

0.5–5.0 1.4% 5.0-150 2.0 % 150-2500 0.9% Reproducibility (RSD R ): No data

• System suitability:

Blank acceptance criteria No peaks detected except that of Norleucine internal standard.

Acceptance of the Calibration curve The calibration curve is considered acceptable if the Linear Regression coefficient is ≥ 0.995 .

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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. But it is necessary 4. Are all the figures and tables pertinent? Yes. 5. Could some be omitted and covered by a simple statement? Yes. It is necessary to describe the SPIFAN samples analyzed and the procedure used to determine the analytical range 6. Are the references complete and correctly annotated? Yes. 7. Does the method contain adequate safety precaution reference and/or statements? Yes, in the method there is a warning about the security measures to be adopted.

Recommendation:

In my opinion, is necessary to make a review of SMPR parameters. It is necessary to describe the SPIFAN samples be analyzed and the procedure used to determine the analytical range. After that method could be recommended to move to First Action .

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