AOAC OMA# 2015.10 Methods Review (January 23, 2019)

AOAC INTERNATIONAL Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN)

NUTRIENTS EXPERT REVIEW PANEL 2015.10 Carnitine/Choline (Methods Review)

January 23, 2019

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

UNITED STATES dboyd@aoac.org 301.924.7077 x126

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method - 2015.10 (Reviewer 1)

Method Title Determination of Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula by Liquid Chromatography/Tandem Mass Spectrometry (LCMSMS)

Method Author Jeffrey Shippar

Reviewer Name: Reviewer 1

Summary of Method Samples are extracted in water for free carnitine and choline. For total carnitine and choline, samples are digested with nitric acid by microwave. Free and total extracts are both diluted to volume with water, mixed with acetonitrile, and analyzed using liquid chromatography (LC) with tandem mass spectrometric (MS/MS) detection.

Method Scope/Applicability Applicable to the measurement of total and free carnitine and choline by UHPLC MS/ MS in infant formula and adult/pediatric nutritional formula.

General Comments about the method Good and simple method employing internal standard for improved accuracy and precision purpose.

Method Clarity Good, could be easily followed by the collaborators.

Pros/Strengths Nicely validated method in most of the aspects. Method capable of analyzing both choline as well as carnitine and both as free as well as total. • The method uses internal std for both analytes during extraction as well as analysis. • Specific for analytes • The method is relatively faster and simple

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Cons/Weaknesses None

General Comment(s) All required supporting data required to establish method, accuracy, repeatability and reproducibility available.

Method Optimization Performed as a part of SLV. The optimization data and validation approved by ERP when the method was approved for first action.

Analytical Range meets SMPR, Part of SLV - First action

LOQ meets SMPR, Part of SLV - First action

Accuracy/Recovery meets SMPR, Part of SLV - First action

Precision (RSDr) Repeatability (RSDr) of free carnitine analysis in all of the MLT samples with its level higher than LOQ was found to be in range of 1.6-3.9% against the SMPR requirement of maximum of 8%. RSDr of MLT samples with its levels > LOQ of total carnitine ranged from 1.8-3.9% across the 8 samples thus far lower than the SMPR target of maximum of 8%. Repeatability of the free choline analysis in MLT samples >2.00mg/100g, was found to be in range of 1.7- 3.7% across these samples thus far lower than the SMPR target of maximum of 5-10%. Total choline analysis in all of the MLT samples gave RSDr values ranging from 1.3-3.0%, far lower than the SMPR requirements of maximum of 5-10%. Reproducibility (RSDR) Reproducibility (RSDR) for free carnitine analysis in all of the MLT samples with its levels higher than LOQ was found to be in range of 2.6-6.3% against the SMPR requirement of maximum of 8%. RSDR of MLT samples with its levels higher than LOQ of total carnitine ranged from 3.7-5.8% across the 8 samples thus far lower than the SMPR target of equal or2.00mg/100g was found to be in range of 4.4- 9.1% across these samples thus lower than the SMPR target of maximum of 5-10%. Total choline analysis in all of the MLT samples except one gave RSDR values ranging from 5.2-9.0% thus meeting the SMPR target of maximum 10-15%. But one of the sample (the adult nutritional ready to feed-high fat sample) provided total choline RSDR value somewhat higher than the SMPR requirements

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(11.1% vs 10%). One of the reasons of this discrepancy according to the author can be that this sample was directed to be used pre-diluted, but not all labs executed this protocol. System Suitability 1. Full calibration curves for carnitine and choline were required to have coefficients of determination (r2) of 0.995 or greater and back-calculated concentrations of the calibration standards not exceed ±10% of theoretical.

2. Signal to noise for the low calibration standard was required to be at least 5:1 for each analyte.

3. Solvent blanks and extracted blanks were required to have

Recommendation The method is recommended for the Final action for Determination of Free and Total Carnitine and Choline in Infant Formula and Adult/Pediatric Nutritional Formula. The method did meet the repeatability as well as the reproducibility requirements of the SMPR in all the MLT samples for the analysis of free and total carnitine as well as free and total choline except one sample which provided RSDR value for total choline of 11.1% compared to the SMPR requirements of maximum of 10%.

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Expert Review Panel for Infant Formula and Adult Nutrition Evaluation of Method - 2015.10 (Reviewer 2)

Title: AOAC Official Method 2015.10 / Free and Total Choline and Carnitine in Infant Formula and Adult/Pediatric Nutritional Formula / Liquid Chromatography / Tandem Mass Spectrometry (LC/MS/MS) First Action 2015 Author: Reviewer Name: Reviewer 2 Summary of Method: Method Scope/Applicability: This review is for the choline portion of the method only. The SMPR for choline simply references “determination of total choline”. Although the method outlines the determination of both free and total forms, and MLT data was included for both free and total forms, the final evaluation for suitability was made strictly against the SMPR requirement for measurement of total choline. General comments about the method: Method Clarity: Method Safety Concerns: Pros/Strengths: •

Cons/Weaknesses •

Supporting Data • General Comment:

Method Optimization:

• Performance Characteristics:

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Analytical Range:

LOQ:

Accuracy/Recovery:

Precision (RSD r ): Reproducibility (RSD R ):

Design of MLT: The study was conducted on ten test materials, one of which was NIST SRM 1849a. After qualification, each participating laboratory analyzed a blind duplicate of each sample for both the free and total portions of each nutrient on each of two days. A total of 9 laboratories produced reportable results.

Choline

SMPR range

2 – 250 mg/100g

RSD r

2 – 20 mg/100g: 20 – 200 mg/100g: 2 – 20 mg/100g: 20 – 200 mg/100g:

<10% < 5% <15% <10%

RSD R

Results for SRM p14 of 2015.10(Carn-07)_MLT_Report_01-2019.pdf

Total Choline Certified Mass Fraction:

1090 ± 110

mg/kg

Table 1.6 Individual laboratory values, mg/100g Corresponding CMF:

98 - 120

mg/100g

A

B

C

D

E

F

G

H

I

107 105

107 106

114 113

105 104

98.1 95.4

105 102

106 103

113 112

100

99

Overall average (Table 1.8): 105 mg/100g;  RSD r = 1.3%; RSD R 5.1%;

 A total of 18 values were used (no outliers); • 17 of the 18 values fell within the CMF.

Total Choline Table 1.8 • 10 of 10 samples produced results in the SMPR range;

3 samples were in the lower SMPR range of 2 – 20 mg/100g  RSD r ranged from 1.5 – 2.7% • The requirement of RSD r < 10% was thus met;  RSD R ranged from 7.2 – 7.5% • The requirement of RSD R < 15% was thus met.

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7 samples were in the upper SMPR range of 20 – 200 mg/100g  RSD r ranged from 1.3 – 3.0% • The requirement of RSD r < 5% was thus met;  RSD R ranged from 5.2 – 11.4%; • The requirement of RSD R

< 10% was met for 6 of 7 samples: o 1 sample in the upper concentration range exhibited an RSD R

value of

11.4%

• System suitability:

Recommendation: Recommend for approval as Final Action for Total Choline.

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