AOAC SPIFAN ERP Meeting Book-March 16, 2016

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

REVIEW TEAM MEETING

December 2015

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

UNITED STATES dboyd@aoac.org 301.924.7077 x126

AOAC INTERNATIONAL Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN) AOAC SPIFAN REVIEW MEETING DRAFT AGENDA

(Method Submissions: Deadline February 3, 2016)

December 2015

1) WELCOME & INTRODUCTIONS (Sullivan)

2) DISCUSS METHOD(S) SUBMITTED

a) Biotin

b) Folate

c) FOS

d) Vitamin K

e) Vitamin C - Reproducibility Testing

3) NEXT STEPS

Conference numbers/code:

1-877-647-3411 US/Canada

Pass code: 774 157 5179#

To view a list of toll-free international dial-in numbers Click Here .

AOAC SPIFAN METHODS SUBMISSION Deadline: February 3, 2016

Date First Name Last Name

Organization

E‐mail Address

Nutrient

Method Title

Method Applicability

Develop and validate a LC‐MS/MS method  for quantitation of vitamin K in infant  formula and adult nutritional samples

Validation of A LC‐MS/MS Method for  Vitamin K Analysis in Infant Formula and  Adult Nutritional Samples Validation of A LC‐MS/MS Method for Folate  Analysis in Infant Formula and Adult  Nutritional Samples (Folate‐22, OMA  2011.06) R&D report 15005n‐MSS‐MSS‐SLV FOS‐01  AOAC 997.08

9/9/2015 Sneh

Bhandari

Silliker

sneh.bhandari@silliker.com

Vitamin K

Infant formula and adult Nutritional  Formulas

9/9/2015 Sneh

Bhandari

Silliker

sneh.bhandari@silliker.com

Folate

SPIFAN FOS‐01 Single Lab Validation for  fructan in Infant Formula and Adult  Nutritionals. The AOAC 997.08 method  was applied for this testing as this method  is one of the 2 AOAC approved methods  for fructan determination

Tiense  Suikerraffinaderij/Be neo‐Orafti

9/17/2015 Monique Steegmans

Monique.Steegmans@raftir.be

Fructans (FOS)

Infant Formula and Adult / Pediatric Nutritional Formulas

DETERMINATION OF BIOTIN BY HPLC‐UV  COUPLED WITH IMMUNOAFFINITY COLUMN  CLEAN‐UP EXTRACTION AOAC First Action Method 2012.22 Ascorbic  Acid (Vitamin C) in Infant Formula and  Adult/Pediatric Nutritionals

9/17/2015 George

Joseph

AsureQuality

George.Joseph@asurequality.com Biotin

Infant Formula and Adult / Pediatric Nutritional Formulas

11/26/2015 Frederic

Martin

Nestle

frederic.martin@rdls.nestle.com Vitamin C

AOAC SMPR 2014.005

Table 1. Method performance requirements a Analytical range

0.1–150 b

Limit of quantitation (LOQ)

≤0.1 b

Standard Method Performance Requirements for Biotin in Infant Formula and Adult/Pediatric Nutritional Formula

Repeatability (RSD r )

0.1–1 b

≤8% ≤6%

>1 b

Recovery

0.1–1 b

80 to 120% of mean spiked recovery over the range of the assay 90 to 110% of mean spiked recovery over the range of the assay

Intended Use: Reference Method for Dispute Resolution 1 Applicability Determination of total biotin in all forms of infant, adult, and/ or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment [AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN); 2010], made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein. d-Biotin.— 5-[(3aS,4S,6aR)-2-oxohexahydrothieno[3,4-d] imidazol-4-yl]pentanoic acid ( see Figure 1). Infant formula .—Breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding (Codex Standard 72-1981) made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein. Limit of detection (LOD) .—The minimum concentration or mass of analyte that can be detected in a given matrix with no greater than 5% false-positive risk and 5% false-negative risk. Limit of quantitation (LOQ) .—The minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. Repeatability .—Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator, and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ).

>1 b

Reproducibility (RSD R )

0.1–1 b

≤16% ≤12%

>1 b

a  Concentrations apply to (a) “ready-to-feed” liquids “as is”; (b) reconstituted powders (25 g into 200 g of water); and (c) liquid

concentrates diluted 1:1 by weight. b  μg/100 g reconstituted final product.

Reproducibility.— The standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility relative standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). Recovery .—The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. 4 Method Performance Requirements See Table 1. 5 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples, and check standards at the lowest point and midrange point of the analytical range. 6 Reference Material(s) National Institute of Standards and Technology (NIST) Standard Reference Material® (SRM) 1849a Infant/Adult Nutritional Formula or equivalent. The SRM is a milk-based, hybrid infant/ adult nutritional powder prepared by a manufacturer of infant formula and adult nutritional products. A unit of SRM 1849a consists of 10 packets, each containing approximately 10 g of material. Certified value of NIST 1849a is 1.99 ± 0.13 mg/kg biotin. 7 Validation Guidance Recommended level of validation: Official Methods of Analysis SM . 8 Maximum Time-to-Result No maximum time. Approved by AOAC Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Final Version Date: March 18, 2014.

Figure 1. d-Biotin.

© 2014 AOAC INTERNATIONAL

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APPENDIX 1

DETERMINATION OF BIOTIN BY HPLC-UV COUPLED WITH IMMUNOAFFINITY COLUMN CLEAN-UP EXTRACTION 1. INTRODUCTION AsureQuality Auckland Laboratory has initiated a method to facilitate a specific, precise, accurate and robust procedure for the analysis of biotin from Infant Formula and Adult / Pediatric Nutritional Formulas. The method also has an assured limit of quantification of 0.1µg/100g (1ppb) based on a simple mathematical relationship between lowest standard and dilution. The method involves immunoaffinity column (R-Biopharm Rhone, EASI- EXTRACT biotin column) clean-up and extraction followed by liquid chromatography with UV detection. The sample is dispersed in phosphate buffered saline and autoclaved at 121±2 ⁰ C for 25 minutes. The sample is cooled to room temperature and then diluted to 100mL in a volumetric flask. The extract is centrifuged and filtered using a Whatman glass microfiber filter paper. Clear filtrate is collected for clean-up and extraction. Biotin immunoaffinity column is mounted onto a SPE manifold. A disposable syringe barrel is connected to the affinity column as a reservoir. The buffer in the affinity column is drained and the sample filtrate is loaded through the reservoir and allowed to flow through by gravity. The column is washed with phosphate buffered saline followed by water. Air is passed through the column to remove residual liquid. Biotin from the column is eluted with methanol and collected in a reacti-vial. The eluent is evaporated to dryness using a heating block set at 85±5°C and the sample is re-constituted in 1mL of water. The biotin in the reconstituted sample is quantified by HPLC using a UV detector set at 200nm. 2. PRINCIPLE / METHODOLOGY

3. CHEMICALS 1. Laboratory Reagent Grade Water

2. Sodium Dihydrogen Phosphate Dihydrate 3. Disodium Hydrogen Phosphate Dihydrate 4. Sodium Hydroxide

5. Methanol, HPLC grade 6. Acetonitrile, HPLC grade 7. Ortho-phosphoric acid, 85% 8. Phosphate buffered saline (PBS) pH 7.4 (Life Technologies 100100.31 or equivalent) 9. Biotin, purity ≥ 99% (Sigma B4501 or equivalent) 4. REAGENTS 1. 2 M Sodium Hydroxide : Weigh 80g of sodium hydroxide into a 1L volumetric flask, dissolve in water and make up to mark. 2. 0.15 M Sodium Phosphate Buffer : Weigh 9.15g of sodium dihydrogen phosphate dihydrate and 16.31g of disodium hydrogen phosphate dihydrate into a 1L volumetric flask. Dissolve in water and make up to mark. Adjust pH to 7 with 2M sodium hydroxide.

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3. 0.1% Phosphoric acid: In a 1L volumetric flask, add 500 mL water. Add 1.2 mL of ortho- phosphoric acid. Mix and make up to mark with water.

5. APPARATUS 1.Whatman Glass Microfiber Filters, CAT No. 1820-125 2.Biopharm Immunoaffinity column Pack P82/ P82B 3.SPE Manifold with accessories 4.Autoclave set at 121 0 C

6. SAMPLE PREPARATION Note: Please see Table 1 for weight and loading volume for different product range. Slurry may be used wherever product heterogeneity is expected. Slurry: Reconstitute the 25g powder with warm water (~50ºC) to a total weight of 200g. Mix thoroughly on a horizontal shaker for 15 minutes and then sonicate at 50 0 C for 10 minutes. Cool to room temperature. 1. Weigh sample/ slurry into a 100mL amber glass screw cap bottle. Refer to Table 1. 2. Add 0.15 M sodium phosphate buffer to a volume of 50mL. 3. Swirl gently to mix. 4. Autoclave the sample preparation at 121 0 C for 25 minutes. 5. Cool the sample to room temperature. Quantitatively transfer the extracts into a 100mL volumetric flask and make up to mark with 0.15 M sodium phosphate buffer. 6. Transfer extracts into centrifuge tubes and centrifuge the samples at 4000rpm for 15min. 7. Filter the samples using the Whatman glass microfiber filter paper (re-filter the samples if the filtrate looks cloudy). 8. Set up the SPE manifold. Attach the immunoaffinity column connected to a 10mL reservoir. Drain off the buffer just above the gel. 9. Load filtrate onto the column and let the solution pass through the column by gravity. 10. Wash the column by passing 10mL of PBS through the column followed by 10mL of water. 11. Introduce a reacti-vial and elute the analyte under gravity with 2mL methanol. Elute further with additional 1mL of methanol. Back flush at least 3 times when eluting. 12. Evaporate the eluent to dryness using a heating block set at 85 + 5 0 C, under a gentle nitrogen blow down. 13. Re-dissolve with 1mL water, cap the reacti-vials and vortex for 30 seconds. Filter and vial for HPLC analysis. Table 1: Sample Preparation Product (µg/100g) Sample Preparation Conc (µg/100mL)

Volume (mL)

Weight (g)

Load (mL)

Final

Min

Max

Min

Max

0.1 1.0 5.0

1.0 5.0

20 10

100 100 100 100 100

50 10 10 10

1 mL 1 mL 1 mL 1 mL 1 mL

1 1 1 5

10

5

50.0

2 (Slurry 16g) 1 (Slurry 8g)

10 10

50.0

100.0 250.0

100.0

0.5

5

2.5

6.25

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7. STANDARD PREPARATION Stock Standard (100µg/mL): Weigh 25mg of biotin reference material into a 250mL amber volumetric flask. Add 150mL of water and shake on an orbital shaker for 24 hours.

Intermediate Standard (100µg/100mL): Dilute 1mL of the stock standard to 100mL with water.

Standard 1 (1.0µg/100mL): Dilute 100µL of intermediate standard to 10mL with water. Standard 2 (2.5µg/100mL): Dilute 250µL of intermediate standard to 10mL with water. Standard 3 (5.0µg/100mL): Dilute 500µL of intermediate standard to 10mL with water. Standard 4 (7.5µg/100mL): Dilute 750µL of intermediate standard to 10mL with water. Standard 5 (10µg/100mL): Dilute 1mL of intermediate standard to 10mL with water.

8. CHROMATOGRAPHIC CONDITIONS 1. Mobile Phase A : 0.1% Phosphoric acid

2. Mobile Pahse B : 100% Acetonitrile 3. Mobile Phase C : 80% Acetonitrile 4. Column: Phenomenex Kinetex Phenyl-Hexyl (150mm x 4.6mm x 2.6µm x 100A) 5. Flow rate: 0.6 mL/min 6. Column Temperature: 25 0 C 7. Run time: 27 minutes 8. Detector: UV diode-array operating at 200nm 9. Injection volume: 100µL

Table 2: Gradient Program

Time (minutes)

Mobile Phase A (%)

Mobile Phase B (%)

Mobile Phase C (%)

0.0

90 90

10 10

0 0

18.0

18.5

0

0

100

24.0

0

0

100

24.5

90

10

0

27.0

90

10

0

9. SENSITIVITY Limit of Quantitation (LOQ) was calculated based on the lowest working standard and the dilution factor. LOQ = (1 x 100) / (20 x 50) = 0.1µg/100g (1ppb) 1 = 1µg/100mL lowest standard 100 = Volume (mL) 20 = 20g sample 50 = Volume (mL) loaded on immunoaffinity column 1= Final volume (mL)

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Protocol Validation Verification

AsureQuality Log Number: 14/05V

Copy Number: 01/01

DETERMINATION OF BIOTIN BY HPLC - UV COUPLED WITH IMMUNOAFFINITY COLUMN CLEAN - UP EXTRACTION

Areas Covered in Validation: Infant and Adult/Paediatric Nutritional Formula

Document Revision Number: 01

AsureQuality

Signature

Printed Name

Job Title

Date

Compiled by

R. Devi

Scientist

18 Sep 2015

Approved by

Printed Name Job Title

Date

Technical

G. Joseph

Technical Manager

18 Sep 2015

Quality Assurance

S. Naidu

QA Analyst

18 Sep 2015

Customer Approval

Signature

Printed Name

Job Title

Date

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REVISION HISTORY

Revision No.

Revision Details

01

Validation report compiled by AsureQuality Auckland Laboratory

DISTRIBUTION RECORD OF CONTROLLED COPIES

Copy No.

Issued To:

Issue Date:

Issued By:

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TABLE OF CONTENTS

Section

Description

Page

1

Introduction

4

2

References

4

3

Scope

4

4

Validation Samples

5

5

Single Laboratory Validation

6

5.1

Methodology

6

5.2

Specificity / Blanks

6

5.3

Identification of biotin

6

5.4

Linearity and range

6

5.5

Accuracy / Recovery

6

5.6

Precision (Repeatability)

7

6

Summary and conclusion

8

Note only include the relevant topics from the table in the report.

Other pages attached:

Yes

No

Appendix 1: Proposed Method Appendix 2: Reagent Blank Appendix 3: Placebo Appendix 4: Linearity and Slope

Appendix 5: Typical Standard Chromatogram Appendix 6: Typical Sample Chromatogram

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1. INTRODUCTION

Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) is an activity mooted by AOAC International and funded by the Infant Nutritional Council of America (INCA). SPIFAN has been founded to develop global standards (dispute resolution methods) for significant nutrients in infant formula and adult nutritionals. AOAC SPIFAN activity launched biotin in August 2013. An AOAC Expert Review Panels will approve one method for Biotin analysis that has undergone single laboratory validation (SLV) as First Action Official Method. The approved method will eventually undergo multi-laboratory testing (MLT) in support of achieving Final Action Official Method status. AsureQuality Auckland Laboratory has initiated a method to facilitate a specific, precise, accurate and robust procedure for the analysis of biotin from Infant Formula and Adult / Pediatric Nutritional Formulas. The method also has an assured limit of quantification of 0.1µg/100g (1ppb) based on a simple mathematical relationship between lowest standard and dilution. The method involves immunoaffinity column (R-Biopharm Rhone, EASI- EXTRACT biotin column) clean-up and extraction followed by liquid chromatography with UV detection. The purpose of this report is to document a single laboratory validation performed as per the AOAC SLV guidelines using SPIFAN matrices. The overall method performance is evaluated using SLV data and is compared to biotin SMPR 2014.005, March 18 2014. 1. AOAC SMPR 2014.005; Final; March 18, 2014: Standard Method Performance Requirements for Biotin in Infant and Adult/Paediatric Nutritional Formula. 2. AOAC Recommended Guidelines for Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Single Laboratory Validation; September 2011. 3. EASI-EXTRACT® Biotin; Immunoaffinity columns for use in conjunction with HPLC or LC-MS/MS, R-Biopharm Rhone Ltd.

2. REFERENCES

3. SCOPE

This validation has been designed to demonstrate the performance of the biotin method by AsureQuality / R-Biopharm Rhone Ltd (Bio-02) and to prove the suitability of the method as a SPIFAN Global standard for infant formula and adult / paediatric nutritional formula as per AOAC SMPR 2014.005, March 18 2014. Specially selected SPIFAN SLV kit materials (matrices) supplied to the laboratory by AOAC International were used for the validation.

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4.VALIDATION SAMPLES

The matrices for the single laboratory validation were supplied by AOAC SPIFAN to AsureQuality’s Auckland laboratory where the SLV was carried out. The description of SLV test materials kit provided for the study is given in Table 1.

Table 1: SLV samples

Container Size (g)

Category

Sample Name

Quantity

Lot # / ID

Child Formula Powder Infant Elemental Powder Adult Nutritional RTF, High Protein Adult Nutritional RTF, High Fat Infant Formula RTF, Milk Based Adult Nutritional Powder Milk Protein Based Infant Formula Powder Partially Hydrolyzed Milk Based Infant Formula Powder Partially Hydrolyzed Soy Based Adult Nutritional Powder Low Fat Child Formula Powder Infant Elemental Powder Infant Formula Powder Milk Based Infant Formula Powder Soy Based SRM 1849a

1

250

00411RF00

1

250

00402RF00

Placebo

1

250

00415RF00

1

250

00407RF00

SPIFAN Blank Milk Form

12

60

6

10

CLC10-b

2

400

11750017V3

2

360

1172572116

2

360

117257651Z

2

250

00394RF00

2

250

00412RF00

Fortified

2

250

00403RF00

2

360

D04HTCVV

2

730

E29JVLV

SPIFAN Control Milk Form

Infant Formula RTF Milk Based

12

60

Adult Nutritional RTF High Protein Adult Nutritional RTF High Fat

3

250

00414RF00

3

250

00406RF00

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5. SINGLE LABORATORY VALIDATION 5.1 Methodology

The sample is dispersed in phosphate buffered saline and autoclaved at 121±2 ⁰ C for 25 minutes. The sample is cooled to room temperature and then diluted to 100mL in a volumetric flask. The extract is centrifuged and filtered using a Whatman glass microfiber filter paper. Clear filtrate is collected for clean-up and extraction. Biotin immunoaffinity column is mounted onto a SPE manifold. A disposable syringe barrel is connected to the affinity column as a reservoir. The buffer in the affinity column is drained and the sample filtrate is loaded through the reservoir and allowed to flow through by gravity. The column is washed with phosphate buffered saline followed by water. Air is passed through the column to remove residual liquid. Biotin from the column is eluted with methanol and collected in a reacti-vial. The eluent is evaporated to dryness using a heating block set at 85±5°C and the sample is re-constituted in 1mL of water . The biotin in the reconstituted sample is quantified by HPLC using a UV detector set at 200nm. Refer to Appendix 1 for detailed methodology

5.2 Specificity / Blanks

Reagent blanks and placebos were taken through the sample preparation procedure and analysed. There was no response (interference) observed in the reagent blanks at the retention time of biotin (Appendix 2). Infant elemental powder showed no response around the retention time of biotin (Appendix 3). However for placebos; child formula powder, adult nutritional RTF high fat and infant formula RTF milk based, a response around the retention time of biotin was detected.

5.3 Identification of biotin

The response (peak) of biotin in the samples was identified by absolute retention time with reference to external standards. Refer to Appendix 5 for typical standard chromatogram and Appendix 6 for a typical sample chromatogram.

5.4 Linearity and range

A linearity check with six levels of working standards (1.0µg/100mL, 2.5µg/100mL, 5.0µg/100mL, 7.5µg/100mL, 10.0µg/100mL and 20.0µg/100mL) was carried out to establish linearity of the method. The range of the working standards covers approximately 0.4% to 400% of the biotin concentration of the infant formula and adult nutritional products in the SLV test materials kit. The correlation coefficients (r 2 ) of all the calibration generated during the validation were not less than 0.998 (Appendix 4).

5.5 Accuracy / Recovery

Accuracy of the method was validated using standard reference material (SRM 1849a), in- house reference material (IRM) and spiked recovery studies. Analysing the samples and comparing the analytical result to the known or added value shows the accuracy of the method.

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The placebos or unfortified SPIFAN matrices were spiked at 50% and 150% of the typical target level. In absence of a placebo, fully fortified product was over-spiked at 50% and 100%. The biotin results in both SRM 1849a and IRM were close to 100% of the respective target values confirming the accuracy of the method (Table 2). All the biotin spiked recoveries were within 95% to 105% of added value confirming accuracy of the method (Table 3). The recoveries well exceeds the SMPR 2014.005 requirements of 90% to 110% in all instances.

Table 2: NIST SRM 1849a and IRM Reference Materials Limits

Biotin (µg/100g)

Day 1 199.97

Day 2 199.65

Day 3 196.50

Day 4

Day 5 Day 6

NIST SRM 1849a

199 ± 13

197.87 198.37 198.42

IRM

26.55 ± 7.7

26.12

27.58

25.36

25.61

25.13

25.39

Table 3: Spiked recovery

Recovery (%)

Spiked Levels

Placebo

Day 1

Day 2

Day 3

Average

50%

99.18 99.40 99.42 94.91 97.98 95.41 97.15 94.87

99.66 98.89 99.94 94.38 98.56 95.25 97.73 95.13

101.00 100.95 99.39 99.72

99.98 98.71 99.63 94.81 98.23 95.40 97.32 95.20

Child Formula Powder

150%

99.28

98.05 98.90 95.33 97.58 94.61 97.50 95.86

99.97 96.65 99.91 99.14 94.47 94.47 99.21 97.85 97.35 94.66 97.18 97.85 95.06 94.77

50%

100.44

Infant Elemental Powder

150%

95.28 98.17 95.11 96.51 95.51

50%

Adult Nutritional RTF, High Fat

150%

50%

Infant Formula RTF, Milk Based

150%

*Infant Formula RTF, Milk Based

50%

97.49

98.07

93.52

94.50

97.51 98.19

96.55

Spiked Levels

Sample

Day 1

Day 2

Day 3

Average

10 3.0 0 96. 25

103. 98 97.6 5

50%

103.50

105.21

103.42 103. 01

103.69

**Adult Nutritional RTF High Protein

95.2 4

100% 96.73 * Calibration forced through origin as the 50% levels were lower than the lowest calibration. **Placebo for Adult Nutritional RTF, High Protein Lot#/ID 00415RF00 could not be used for spiked recovery studies due to the bacterial fermentation of milk. Instead the fully fortified sample of the same matrix Lot#/ID00414RF00 was used for over spiking. 96.95 97.04 97.24

5.6 Precision (Repeatability)

Repeatability was determined by analysing the 12 different matrices in the SLV test materials kit in duplicate on each of the 6 days by two different analysts. In addition two different liquid chromatography equipment were utilized during the course of validation. Precision data for all the samples can be found in Table 4. Coefficient of variation (%RSD) was calculated on all the 12 data points on all the 12 SLV matrices. The %RSD was not more than 3.2% and is well below the set limit of 6% as per SMPR 2014.005.

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Table 4: Repeatability

Biotin (µg/100g)

SPIFAN SLV Samples

RSD

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Average

CLC10-b 197.42 201.01 196.08 196.28 197.93 201.54 198.54 198.39 199.34 198.81 197.00 197.30 198.30 0.86 11750017V3 12.77 12.73 12.67 12.77 12.69 12.69 12.53 12.73 12.61 12.62 12.65 12.70 12.68 0.55 1172572116 34.68 34.50 34.20 35.24 35.17 35.13 36.05 35.09 36.19 36.97 35.19 35.02 35.29 2.19 117257651Z 39.76 39.99 39.98 39.16 40.12 40.50 39.37 40.77 40.87 40.96 39.50 40.70 40.14 1.54 00394RF00 248.67 249.85 254.88 237.61 258.75 261.19 256.65 259.83 255.74 259.20 254.28 256.30 254.41 2.55 00412RF00 91.08 90.66 88.99 89.01 89.70 91.71 89.92 90.81 89.56 90.20 90.69 90.72 90.25 0.93 00403RF00 69.17 69.30 70.41 69.88 71.88 72.28 71.35 71.45 72.14 71.83 70.52 71.03 70.94 1.52 D04HTCVV 35.48 35.24 34.23 33.92 33.36 34.28 33.01 33.17 32.94 32.96 31.71 32.94 33.60 3.19 E29JVLV 43.30 43.25 45.40 44.72 45.02 44.38 45.29 44.85 45.15 44.78 44.76 45.08 44.67 1.58 Control Milk F 5.45 5.47 5.36 5.34 5.40 5.44 5.38 5.43 5.20 5.31 5.15 5.37 5.36 1.83 00414RF00 54.02 55.42 55.50 55.16 52.36 52.07 53.67 53.78 53.23 53.91 55.48 54.21 54.07 2.16 00406RF00 68.88 65.17 63.77 64.85 66.78 67.48 65.51 66.54 65.64 65.36 63.92 62.13 65.50 2.74 SRM 1849a 198.46 0.63 199.97 199.65 196.50 197.87 198.37 198.42 AsureQuality Auckland Laboratory has initiated a method to facilitate a specific, precise, accurate and robust procedure for the analysis of biotin from Infant Formula and Adult / Pediatric Nutritional Formulas. The method involves immunoaffinity column (R-Biopharm Rhone, EASI-EXTRACT biotin column) clean-up and extraction followed by liquid chromatography with UV detection. The proposed method exceeds all the SPIFAN SMPR for biotin. The method also has an assured limit of quantification of 0.1µg/100g (1ppb) based on a simple mathematical relationship between lowest standard and dilution. This method is found to be suitable for routine testing of biotin in infant formula and adult nutritionals. Comparison of SMPR for biotin and the proposed method performance are given in Table 5. 6. SUMMARY AND CONCLUSION

Table 5: Performance of the proposed method (Bio -02) compared to SMPR 2014.005 Parameters SMPR 2014.005 Proposed Method Comments

Analytical range of the proposed method is wider based on the sample weight taken and loading volume for sample extraction. The proposed method meets the LOQ of 0.1 µg/100g as required by AOAC SMPR 2014.005.

Analytical Range

0.1 - 150 µg/100g

0.1 - 300 µg/100g

Limit of Quantitation

≤ 0.1 µg/100g

≤ 0.1 µg/100g

Repeatability of the proposed method is better than the SMPR 2014.005.

Repeatability (RSD r )

>1 µg/100g: ≤ 6%

>1 µg/100g: ≤ 4%

All the spiked recoveries by the proposed method are better than the recovery range specified by AOAC SMPR 2014.005.

Recovery (>1µg/100g)

90 to 110%

95 to 105%

NIST 1949a results are at the certified value.

NIST SRM 1849a

199 ± 13

198 ± 3

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AOAC SMPR 2011.006

Table 1. Method performance requirements a Analytical range 0.50–300 b Limit of detection (LOD) ≤0.10 b Limit of quantitation (LOQ) ≤0.50 b Repeatability (RSD r ) 0.50 b

Standard Method Performance Requirements for Folate in Infant Formula and Adult/Pediatric Nutritional Formula

≤11%

21.5 b 43.0 b 64.0 b 85.0 b 21.5 b 43.0 b 64.0 b 85.0 b 0.5 21.5 b 43.0 b 64.0 b 85.0 b 0.5 b

Intended Use: Global Dispute Resolution Method

≤7%

1 Applicability Determination of total folate [supplemental folic acid (CAS 59- 30-3) or 5-methyl-tetrahydrofolate (CAS 68792-52-9), and endogenous 5-methyl-tetrahydrofolate polyglutamate] in all forms (powders, ready-to-feed liquids, and liquid concentrates) of infant, adult, and pediatric nutritional formula. 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment (AOAC SPIFAN, 2010), made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein. Infant formula .—Breast-milk substitute specially manufactured to satisfy, by itself, the nutritional requirements of infants during the first months of life up to the introduction of appropriate complementary feeding (Codex Standard 72-1981), made from any combination of milk, soy, rice, whey, hydrolyzed protein, starch, and amino acids, with and without intact protein. Limit of detection (LOD) .—The minimum concentration or mass of analyte that can be detected in a given matrix with no greater than 5% false-positive risk and 5% false-negative risk. Limit of quantitation (LOQ) .—The minimum concentration or mass of analyte in a given matrix that can be reported as a quantitative result. Repeatability .—Variation arising when all efforts are made to keep conditions constant by using the same instrument and operator, and repeating during a short time period. Expressed as the repeatability standard deviation (SD r ); or % repeatability relative standard deviation (%RSD r ). Reproducibility .—The SD or RSD calculated from among- laboratory data; expressed as the reproducibility standard deviation (SD R ), or % reproducibility relative standard deviation (%RSD R ). Recovery .—The fraction or percentage of spiked analyte that is recovered when the test sample is analyzed using the entire method. 4 Method Performance Requirements See Table 1. 5 System Suitability Tests and/or Analytical Quality Control Suitable methods will include blank check samples and check standards at the lowest point and midrange point of the analytical range. 6 Reference Material(s) National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1849 Infant/Adult Nutritional Formula,

Recovery

90–110%

Reproducibility (RSD R )

≤32%

≤16%

a Concentrations apply to ( 1 ) “ready-to-feed” liquids “as is”; ( 2 ) reconstituted powders (25 g into 200 g water); and ( 3 ) liquid concentrates diluted 1:1 by weight. b μg/100 g expressed as folic acid in reconstituted final product.

or equivalent. The SRM is a milk-based, hybrid infant/adult nutritional powder prepared by a manufacturer of infant formula and adult nutritional products. A unit of SRM 1849 consists of 10 packets, each containing approximately 10 g of material. Certified value of folic acid in NIST 1849 is 2.11 (±0.13) mg/kg. Note : The reference value for NIST 1849 is defined in terms of folic acid. The performance parameters in this SMPR are intended for folate and 5-methyl-tetrahydrofolate polyglutamate. Some discrepancy may be expected. 7 Validation Guidance Recommended level of validation: Official Methods of Analysis SM . 8 Maximum Time-to-Signal No maximum time. Approved by Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Final Version Date: April 5, 2011. Effective Date: June 29, 2011.

© 2012 AOAC INTERNATIONAL

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Silliker, Inc. Chemistry Research Center Report RPN 15-002

Validation of A LC-MS/MS Method for Folate Analysis in Infant Formula and Adult Nutritional Samples (Folate-22, OMA 2011.06)

Prepared for: AOAC International

Prepared by: Sneh D. Bhandari, Ph.D.. John Szpylka, Ph.D. Director, Chemistry R&D 3600 Eagle Nest Drive Crete, IL 60417 sneh.Bhandari@silliker.com john.szpylka@silliker.com

Date : 8th September, 2015

The entire content of this REPORT is subject to copyright protection. Copyright © 2015 Silliker, Inc. All rights reserved. The contents of this REPORT may not be copied other than for use by non-for-profit organization, and appropriate reference with all copyright notices stated. The REPORT may not be copied, reproduced or otherwise redistributed. Except as expressly provided above, copying, displaying, downloading, distributing, modifying, reproducing, republishing or retransmitting any information, text or documents contained in this REPORT or any portion thereof in any electronic medium or in hard copy, or creating any derivative work based on such documents, is prohibited without the express written consent of Silliker, Inc. Nothing contained herein shall be construed as conferring by implication, estoppel or otherwise any license or right under any copyright of Silliker,

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Validation of A LC-MS/MS Method for Folate Analysis in i nfant Formula and Adult Nutritional Samples

Objective:

Develop and validate a LC-MS/MS method for quantitation of folate in infant formula and adult nutritional samples

Analyte Scope:

Folic acid (FA), , 5-methyl-5,6.7,8-tetrahydrofolate (5-CH3-THF), 5-formyl-5,6.7,8-tetrahydrofolate (5-CHO-THF), 5,6.7,8-tetrahydrofolate (THF), 5,10 methenyl--5,6.7,8-tetrahydrofolate (5,10-CH=CH- THF), 10-methyl-folic acid (CH3-FA), 10-formyl-folic acid (CHO-FA).

Matrix Scope:

Infant Formula Samples Adult Nutritional

SPIFAN Samples Used for the Method Validation in this study

Provided to Silliker by SPIFAN on 3/26/14 1.) Child Formula Powder Lot # 00411RF00 2.) Infant Elemental Powder Lot # 00402RF00 3.) Adult Nutritional RTF, High Protein Lot # 00415RF00 4.) Adult Nutritional RTF, High Fat Lot # 00407RF00 5.) Infant Formula RTF, Milk Based SPIFAN Blank Milk Form The above blank samples are used for the evaluation of spike recovery of folates by the method 6.) SRM 1849A Lot # CLC10-b 7.) Adult Nutritional Powder Milk Protein Based Lot # 11750017V3 8.) Infant Formula Powder Partially Hydrolyzed Milk Based

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Lot # 1172572116 9.) Infant Formula Powder Partially Hydrolyzed Soy Based Lot # 117257651Z 10.) Adult Nutritional Powder Low Fat Lot # 00394RF00 11.) Child Formula Powder Lot # 00412RF00 12.) Infant Elemental Powder Lot # 00403RF00 13.) Infant Formula Powder Milk Based Lot # D04HTCVV 14.) Infant Formula Powder Soy Based Lot # E29JVLV 15.) Infant Formula RTF Milk Based SPIFAN Control Milk Form 16.) Adult Nutritional RTF High Protein Lot # 00414RF00 17.) Adult Nutritional RTF High Fat Lot # 00406RF00 The last 12 samples have been used for the method precision evaluation

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Detailed Method:

Folate Analysis in Food, dietary supplements, feed and pet food samples

Definition: The described method is an UPLC-MS/MS method for determining total folates, and important folate vitamers in various food, dietary supplement, feed and pet food matrices after trienzyme digestion. Rat plasma conjugase is used in deconjugating various folate polyglutamates to folate monoglutamates after protease and α-amylase digestion of the food matrix in the trienzyme digestion process. Scope: This method is applicable to all various food, dietary supplement, feed and pet food matrices.

Analyte Scope:

Table 1: Analyte Scope of the Method

Formula Weight

Precursors [M+H] +

Product Ions

Collision Energy

Folate Vitamers

FA

441 459 473 445 456 455 469

442 460 474 446 457 456 470

295 313 327 299 412 309 295

20 22 20 20 27 20 35

5-CH3-THF 5-CHO-THF 5 ,6,7,8-THF 10-CH3-FA 10-CHO-FA Total Folate

5,10-CH=CH-THF

Range:

The method is linear in the range of 10 μg/100g to 19100 μg/100g total folates. With appropriate adjustment of sample weight and SPE clean up loading volume (Table 1), the range can be extended from 5 μg/100g to 2,000,000 μg/100g total folates in the food matrices and vitamin concentrates described above. Principle: An aliquot of a thoroughly blended mixed sample is accurately is taken for the analysis. Appropriate amount of internal standard and phosphate buffer, pH 6 is added. The sample is extracted a pH 6 buffer containing different folate stabilizers and labeled folic acid and methyl –THF as internal standards by triple enzyme digestion. Rat plasma conjugase is used to de-conjugate the polyglutamate forms of folates to mono-glutamates. The clear extract is then subjected to a weak anion exchange (WAX) SPE . The folates are eluted from SPE and analyzed by UPLC-MS/MS. Seven folate vitamers are quantified and the total folates determined and expressed as folic acid. Isotopically labeled folic acid ( 13 C-Folic acid) is used as the internal standard.

Chemicals:

1.) Folic Acid (Standard) Schircks Laboratories 2.) (6R,S)-5-Methyl-5,6,7,8- Schircks Laboratories Tetrahydrofolic acid, calcium salt 3.) (6S)-5,6,7,8- Tetrahydrofolic Schircks Laboratories

Cat. No. 16.203

Cat. No. 16.235

Cat. No. 16.207

acid 4.) 7,8-Dihydrofolic acid 5.) (6S)-5-Formyl-5,6,7,8-

Schircks Laboratories

Cat. No. 16.206

Schircks Laboratories

Cat. No. 16.221

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

Tetrahydrofolic acid, calcium salt 6.) 10-Methylfolic acid 7.) (6R,S)-5,10-Methylene-5,6,7,8- Tetrahydrofolic acid, calcium salt 8.) 10-Formylfolic acid

Schircks Laboratories

Cat. No. 16.211

Schircks Laboratories

Cat. No. 16.226

Schircks Laboratories

Cat. No. 16.215

9.) Pteroyltri-y-L-glutamic acid Schircks Laboratories (Folic acid, triglutamate) 10.) (6R,S)-5(Formyl-13C)-5,6,7,8- Schircks Laboratories

Cat. No. 16.253

Cat. No. 9622

Tetrahydrofolic acid, lithium salt 11.) (5-15N)-Folic Acid

Schircks Laboratories

Cat. No. 96.203

12.) 13 C-Folic Acid (Internal Standard) Merck Cat. No. CH2QC 900824 13) 13 C5-labeled (6S)-5-methyltetrahydrofolic acid, calcium salt (Internal Std) ((6S)-5-CH3-H4Pte[13C5]Glu-Ca) Merck Cat No. CH2QC 900827 14.) a-amylase Sigma-Aldrich Cat. No. A6211 15.) protease from bacillus Sigma-Aldrich Cat. No. P3910 16.) Conjugase (Rat Plasma) Bioreclamation LLC Cat. No. RATPLLIHP-M 17.) Ammonium Hydroxide Fisher Cat. No. A669-500 18.) Sodium Phosphate, dibasic salt Fisher Cat. No. S-374-500 19.) Methanol, Optima LC/MS, 99.9% Fisher Cat. No. A456-4 20.) Acetic Acid Fisher Cat. No. A38S-212 21.) Sodium Hydroxide Fisher Cat. No. S318-1 22.) Ammonium Formate Sigma-Aldrich Cat. No. 17843-250G 23.) Ammonium Acetate Fisher Cat. No. AX1220-500G 24.) 2-Mercaptoethanol Fisher Cat. No. BP176-100 25.) Ascorbic Acid Fisher Cat. No. BP351-500 26.) Water, Optima Fisher Cat. No. W7-4 27.) TCEP Fisher Cat. No. 50-093-0 28.) Charcoal Fisher Cat. No. D127-500 29) Ammonium hydroxide Fisher Cat. No. A669-500 (Ammonia soln. 28.87%)

Apparatus:

1.) Shimadzu UFLC system 2.) API 3200 Mass Spectrometer 3.) Mettler Toledo pH Meter 4.) 24-well SPE Vacuum manifold 5.) Refrigerator (capable of cooling to 4 o C) 6.) Freezer (capable of cooling to -20 o C) 7.) Water Bath, with shaker 8.) Centrifuge (capable of up to 2000rpm) 9.) Microfuge (capable of up to 15000rpm)

Supplies:

1.) Disposable Glass Culture Tubes Fisher

Cat. No. 14-961-30

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

16x125mm 2.) 50mL centrifuge tubes 3.) PVDF syringe filters 4.) Microcentrifuge tubes; 2mL

Fisher

Cat. No. 14-375-150

Fisher cat. No. 02-681-344 5.) SPE cartridge: Phenomenex Part# 8B-S038UBJ. A mix mode weak anion exchange and reverse phase solid-phase extraction cartridge

Reagents:

1.) Folate Stock Solution Solvent (15.6 mM ammonium acetate buffer with 25% ascorbic acid and 1% mercaptoethanol, pH = 5.5): a.) weigh accurately 1.27g of ammonium acetate and transfer into a 1000mL beaker. b.) In a fume hood, add approximately 700ml of DI water and 4.8 mL of glacial acetic acid slowly. c.) Add 250g of ascorbic acid and 10mL of 2-mercaptoethanol. Stir to completely dissolve using a stir bar. Add concentrated ammonium hydroxide drop wise as necessary to dissolve completely. d.) Check the pH of the solution and adjust to pH 5.5, if necessary, using concentrated ammonium hydroxide or glacial acetic acid. e.) Transfer the solution into a 1L volumetric flask and bring to volume with DI water. 2.) Folate Intermediate Standard Solution Solvent (1.6 mMAmmoniumAcetate buffer, with 1% ascorbic acid, pH 5.5): a.) Weigh accurately 0.12 g ammonium acetate and transfer into a 1000mL beaker. b.) In a fume hood, add approximately 700ml of DI water and 0.5081g (484 uL)of glacial acetic acid slowly. Add 10g of ascorbic acid and stir until completely dissolved. c.) Adjust pH to 5.5 if necessary with concentrated ammonium hydroxide or glacial acetic acid. d.) Transfer the solution to a 1L volumetric flask and bring to volume with DI water. 3.) Solvent for SPE Elution and Folates Standard (Methanol with 10% formic acid and 1% ascorbic acid): a.) Into a 100mL volumetric flask, transfer 10mL of formic acid and 1g of ascorbic acid. Add approximately 50ml methanol. b.) Sonicate in a water bath set at approximately 25 o C until complete dissolution. c.) Bring to volume with methanol. Use solution to elute folates from SPE sorbent after clean up, to make analytical standard of folates and as a blank in UPLC-MS-MS analysis. 4.) Folate Stock Solution (500ug/mL) a.) Accurately weigh out about 5mg of each standard in a separate 10mL low-actinic volumetric flask. b.) Add approximately 7.5mL of the Folates Stock Solution Solvent to each flask and sonicate in a bath set at 40 o C for about 20min or until complete dissolution. Add several drops Ammonium hydroxide to aid in dissolution if necessary. c.) Bring to volume with Folates Stock Solution Solvent and transfer to freezer safe containers. d.) The Folic acid, 13 C-Folic Acid (IS), (6S)-5-Formyl-5,6,7,8-Tetrahydrofolate, 10-methyl- folic acid and 10-formyl-folic acid solutions are stable for up to 180 days stored in the freezer at -70 o C. e.) The (6S,R)-5-Methyl-5,6,7,8-Tetrahydrofolate, and 5,6,7,8-Tetrahydrofolate solutions are

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

stable for only 90 days stored in the freezer at -70 o C. f.) Pteroyltri-y-glutamate (FA-glu 3

) is stable for 12 months stored in the freezer at -70 o C.

Preparation of Stock standards of different folate compounds

Table 2: Preparation of Stock standard solns. (examples) of different folate compounds

Folate compd #

Final Vol* (mL)

Concen (mcg/mL)

Standard

Mass (g)

Shircks (6S)-5-Formyl-5,6,7,8- tetrahydrofolic acid

1

0.00607

10

607

2

10-Formylfolic acid

0.00597

10

597

Shircks (6R,S)-5-methyl-5,6,7,8- etrahydrofolic acid

3

0.00584

10

584

4

10-methylfolic acid

0.00529

10

529

Shircks (6R,S)-5,10-methenyl- 5,6,7,8-tetrahydrofolic acid

5

0.00587

10

587

6**

pteroyltri-y-L-glutamic acid

0.00911

10

911

Shircks (6S)-5,6,7,8-tetrahydrofolic acid

7

0.00542

10

542

8

Shircks Folic acid

0.00555

10

555

* make up volume to 10 mL with stock solution solvent. **used only for checking conjugase activity in rat plasma

5). Folate Internal std.Stock solutions:

a. 13 C-Folic Acid Internal Standard Stock soln. (2000 ug/mL) - The labeled Folic acid, 13 C-Folic Acid Millipore / Merck CH2QC-900824. The chemical is usually bought in 20 mg amount. The entire 20 mg amount of labeled folic acid is dissolved in 10 mL of folate stock std. solvent. Dissolve the chemical completely with the aid of vortex and brief (15 second) sonication. The solution is anticipated to be stable for up to 5 months at -70 o C. Stability to be checked during and after this period before use. b. 13 C5-(6S)-5-Methyl-5,6,7,8-Tetrahydrofolate Internal Standard Stock soln. (2000 ug/mL) : The labeled methyl-THF , 13 C5-(6S)-5-Methyl-5,6,7,8- Tetrahydrofolate (Millipore / Merck CH2QA-900827). The chemical is usually bought in 20 mg amount. The entire 20 mg amount of labeled methyl-THF is dissolved in 10 mL of folate stock std. solvent. Dissolve the chemical completely with the aid of vortex and brief (15 second) sonication. The solution is anticipated to be stable for up to 3 months at -70 o C. Stability to be checked during and after this period before use

6. Folate Intermediate Standard Solution (20ug/mL):

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Silliker Chem, Res. Center Crete, IL – Report of A Validation of LC-MS/MS Method for Folate Analysis

a.) Pipette and appropriate amount of each folate stock solution (EXCPET THE INTERNAL STANDARD STOCK SOLUTION AND PTEROLYTRI-y-GLUTAMIC ACID) into a 10mL low-actinic volumetric flask. b.) Three separate solutions (one mixture and two individual solutions) are prepared as detailed in the following table. c.) Add approximately 5mL of Folate Intermediate Standard Solution Solvent and sonicate in water bath set at 40 o C for about 10min to thoroughly mix. d.) Bring to volume with Folate Intermediate Standard Solvent and store approximately -20 o C. Stable for up to 30days.

Table 3: Preparation intermediate standard solutions

Mixture Intermed or Individual std. Soln

Stock std. Concn (mcg/mL)

Stock std. Volume (uL)

Intermed std Soln #

Folate compd #

Final Volume* (mL)

Concen (mcg/mL)

Stock Standard

Shircks (6S)-5-Formyl-5,6,7,8- tetrahydrofolic acid

1

607

356

21.6092

2

10-Formylfolic acid

597

335

19.9995

Mixture Intermed Std. Soln

4

10-methylfolic acid

529

378

19.9962

I

10

Shircks (6R,S)-5,10-methenyl- 5,6,7,8-tetrahydrofolic acid

5

587

368

21.6016

Shircks (6S)-5,6,7,8- tetrahydrofolic acid

7

542

369

19.9998

Indiv. Intermed. Std soln Indiv. Intermed. Std soln Conjugase check soln

Shircks (6R,S)-5-methyl-5,6,7,8- tetrahydrofolic acid

II

3

584

371

10

21.6664

III

8

Shircks Folic acid

555

360

10

19.98

Conjugase check soln

6

pteroyltri-y-L-glutamic acid

911

222

10

20.2242

*Make up final volume to 10 mL with Folate Intermediate std. solvent.

7.) Internal Standard Intermediate Standard Solution (20ug/mL):

8