2015.14 Vitamin B (July 2018)

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 , and B 6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS

AOAC Final Action 2015.14 Multi Laboratory Study

Final Report

July 24, 2018

Study Directors:

Sean McClure & Louis Salvati

Abbott Nutrition, Columbus Ohio USA

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Study Information

Study Title: Collaborative Study of AOAC First Action 2015.14 for the Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 , and B 6 in Infant Formula and Related Nutritionals by Enzymatic Digestion and LC-MS/MS.

Study Objective: The objective of this study is to complete validation of AOAC First Action 2015.14 for the quantification of total vitamins B 1 , B 2 , B 3 , and B 6 in infant formula and related nutritionals by determining method reproducibility and repeatability. Study Directors Louis Salvati

Abbott Nutrition 3300 Stelzer Road Columbus, OH 43219 P: 614 624-3971 louis.salvati@abbott.com Sean McClure Abbott Nutrition 3300 Stelzer Road Columbus, OH 43219 P: 614 624-5298 sean.mcclure@abbott.com

Study Monitors: AOAC International (SPIFAN) Official Method Board or delegated personnel. MLT Overview Participating laboratories were asked to set up the method (Appendix A), analyze two practice samples in duplicate, and report the results to the study directors. After successful analysis of the practice samples, study participants were asked to analyze 15 fortified and unfortified SPIFAN matrices in duplicate. The samples were split into two groups of 15 per the data reporting templates, and each group was analyzed on a separate day. Participants were asked to reconstitute all powder samples by diluting either 10 or 25 grams of powder to 100 or 225 grams respectively with water. Electronic excel templates for reporting sample weights and results were provided. After completion of the study, the data were analyzed using the AOAC International Interlaboratory Study Workbook for Blind (unpaired) Replicates, Version 2.1 to calculate repeatability standard deviation, s(r), reproducibility standard deviation, s(R), repeatability relative standard deviation, RSD(r), reproducibility relative standard deviation, RSD(R), and HORRAT value and to detect outliers.

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Table 1: MLT Sample Matrices with Codes Product Description

Blind Duplicate Codes

Batch

K16NTAV E10NWZC

CULF358 TJHR217 KDOX966 SWUO667 DYLB360 FPTE312 EFXN778 NSRB999 XKIP216 TJMN542 KGSZ273 RQXQ518 LYNY751 ECHL425 URTF231

GBZC169 OACN211 ATAN351 MYHK654 ZMQM883 DOMY545 BFAO941 JSDT587 HYJU890 XJDD334 LTCT316 GVPE615 PZGP859 UOPM297 WKHN288

Milk-based Infant Formula Powder Soy-based Infant Formula Powder

410057652Z 410457651Z 00729RF00 00730RF00 4052755861 4044755861

Milk-based Partially Hydrolyzed Infant Formula Powder Soy-based Partially Hydrolyzed Infant Formula Powder Adult Nutritional Ready-to-feed- High Fat Adult Nutritional Ready-to-feed- High Protein Milk-based Child Formula Powder Progress Gold Milk-based Infant Formula Stage 1 Powder Illuma Milk-based Infant Formula Ready-to-feed Milk-based Infant Formula Ready-to-feed Placebo

EV4H2R EV4H2Q CLC10-B

SRM 1849a a

00866RF00 00859RF00 00795RF00 50350017W1

Milk-based Child Formula Powder Adult Low Fat Nutritional Powder Infant Elemental Formula Powder FOS/GOS-based Infant Formula Powder

Table 2: AOAC 2015.09 MLT Participating Laboratories Laboratory Address

Country Singapore

Abbott Laboratories

20 Biopolis Way, #09-01/02, Centros Building, Singapore 138668

SHCIQ

1208 Minsheng Rd, Pudong Xinqu, Shanghai Shi, China, 200134 China

Laboratoire AQUANAL 151 bis, avenue Jean Jaures, 33600 PESSAC

France

ACNA

60 Eighth Street, N.E.Atlanta, GA 30309 Block: S-15, Biocon Park, Plot No. 2&3 Bommasandra IV Phase, Jigani Link Road Bangalore 560 100, Karnataka, India

USA

Syngene International Limited

India

Covance EU

Otley Road, Harrogate HG3 1PY

United Kingdom Vietnam

NIFC

65 Phạm Thận Duật, Mai Dịch, Cầu Giấy, Hà Nội P.O. Box 844, Utrechtseweg 48, 3700 AV Zeist

Triskelion

Netherlands

Abbott Laboratories

3300 Stelzer Rd, Columbus OH 43219 Runderweg 6, 8219 PK Lelystad

USA

CCIC

Netherlands

Results and Discussion

Five of the thirteen laboratories that originally signed up to participate submitted full sets of data. Five of the nine labs that agreed to participate at the AOAC mid-year 2018 meeting did submit data in time for this report. Two labs are still working on data and pending.

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Thiamine MLT data are summarized in the tables below.

Table 3: AOAC 2015.14 Thiamine Data (µg/100g) from Analysis of Blind Duplicates, Complete Set of Individual Results

Lab

K16NTAV

E10NWZC

410057652Z

410457651Z

00729RF00

00730RF00

4052755861

1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9

1.24 1.21 1.17 1.26 1.08 1.15 1.10 1.07 1.16 1.15 1.42 1.03 1.12

1.24 1.16 1.22 1.25 1.15 1.15 1.10 1.08 1.13

1.21 1.14 1.13 1.23 1.02 1.07 1.15 1.06 1.00 1.12

1.21 1.14 1.12 1.28 1.01 1.11 1.12 1.08 1.06 1.08

0.871 0.948 0.787 0.811 0.567 0.735 0.829 0.834 0.655 0.801 0.005 0.002 0.003 0.001 0.003 0.000 0.002 0.003 0.003 0.004

0.859

0.855 0.817 0.801 0.858 0.709 0.763 0.725 0.775 0.735 0.782

0.853 0.270 0.276 0.231 0.229 0.530 0.510

1.10

0.792 0.484 0.559 0.488

1.72

0.441 0.433

0.790 0.813 0.566 0.682 0.865 0.828 0.657 0.805 0.004 0.001 0.003 0.000 0.003 0.000 0.002 0.003 0.002 0.004

0.795 0.231 0.231 0.201 0.199 0.505 0.491 0.874 0.238 0.232 0.206 0.224 0.449 0.456 0.719 0.190 0.196 0.159 0.159 0.402 0.408 0.755 0.156 0.158 0.148 0.148 0.423 0.431 0.726 0.155 0.157 0.157 0.150 0.456 0.439 0.743 0.184 0.179 0.177 0.166 0.451 0.465 0.750 0.142 0.140 0.143 0.147 0.428 0.403 0.787 0.164 0.159 0.156 0.154 0.452 0.443

0.936 0.919

10

Lab

4044755861

EV4H2R

EV4H2Q

CLC10-B

00866RF00

00859RF00

00795RF00

1.160 0.149 1.052 1.155

0.0449 0.0395 0.0400 0.0420 0.0320 0.0303 0.0311 0.0356 0.0284

0.0475 0.0357 0.0410 0.0440 0.0310 0.0319 0.0313 0.0321 0.0274

1.47 1.48 1.37 1.43 1.18 1.35 1.30 1.33 1.34 1.31

1.56 1.66 1.31 1.44 1.19 1.30 1.34 1.33 1.25 1.43

3.44 3.64 3.12 3.39 2.67 2.93 3.40 3.45 2.76 3.15

3.45 3.87 3.10 3.31 2.67 2.95 3.55 3.43 2.85 3.19

2.11 1.94 1.96 2.03 1.77 1.84 1.86 1.87 1.88 1.91

2.04 1.91 2.03 2.07 1.78 1.81 1.84 1.89 1.78 2.15

1.79 1.81 1.63 1.75 1.51 1.49 1.61 1.63 1.61 1.63

1.86 1.77 1.59 1.83 1.53 1.56 1.60 1.53 1.63 1.61

0.907 0.913 0.953 0.996

1.04 1.08

1.097 1.097

0.907 0.898

10

1.03

1.05

0.031

0.032

Lab

50350017W1 0.675 0.684 0.620 0.624 0.633 0.628 0.661 0.667 0.506 0.509 0.574 0.566 0.585 0.603 0.608 0.602 0.577 0.561 0.619 0.589

10

HIGHLIGHTED RED ARE RESULTS THAT WERE FLAGGED BY THE COCHRAN’S OR SINGLE/DOUBLE GRUBBS TEST AND REMOVED

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Table 4: Statistical Analysis of MLT Blind Duplicates for Thiamine

Sample ID:

K16NTAV

E10NWZC

410057652Z 410057652Z 410457651Z

00729RF00

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

10 20

10 20

9

10 20

10 20

18

1.1390 0.0217 0.0952

1.1173 0.0231 0.0759

0.7904 0.0378 0.1282

0.7642 0.0154 0.1005

0.7807 0.0108 0.0517

0.2250 0.0171 0.1132

Repeatability RSD, % Reproducibility RSD, %

1.90 8.36 0.27

2.07 6.80 0.22

4.78

2.01

1.39 6.62 0.20

7.61

16.22

13.15

50.29

HORRAT value

0.49

0.39

1.26

Sample ID:

00729RF00

00730RF00

00730RF00

4052755861 4044755861

4044755861

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

9

10 20

9

10 20

10 20

9

18

18

18

0.1920 0.0030 0.0450

0.2680 0.2753 0.3535 102.70 131.90

0.1752 0.0054 0.0319

0.4507 0.0099 0.0360

1.0098 0.2852 0.2852

1.0348 0.0211 0.0919

Repeatability RSD, % Reproducibility RSD, %

1.57

3.08

2.20 7.99 0.22

28.24 28.24

2.04 8.88 0.28

23.41

18.22

HORRAT value

0.57

3.38

0.44

0.88

Sample ID:

EV4H2R

EV4H2Q

CLC10-B

00866RF00

00859RF00

00795RF00

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

10 20

10 20

10 20

10 20

10 20

0.0354 0.0015 0.0062

0.0024 0.0003 0.0015

1.3680 0.0602 0.1209

3.2149 0.0682 0.3430

1.9232 0.0652 0.1156

1.6490 0.0394 0.1157

Repeatability RSD, % Reproducibility RSD, %

4.22

12.63 62.37

4.40 8.84 0.29

2.12

3.39 6.01 0.21

2.39 7.02 0.24

17.39

10.67

HORRAT value

0.33

0.79

0.40

Sample ID:

50350017W1

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

0.6045 0.0092 0.0500

Repeatability RSD, % Reproducibility RSD, %

1.53 8.28 0.24

HORRAT value

In Red are statistics after flagged results were removed

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Riboflavin MLT data are summarized in the tables below.

Table 5: AOAC 2015.14 Riboflavin Data (µg/100g) from Analysis of Blind Duplicates, Complete Set of Individual Results Lab K16NTAV E10NWZC 410057652Z 410457651Z 00729RF00 00730RF00 4052755861 1 1.85 1.59 1.51 0.927 1.92 1.37 0.778 0.976 0.612 0.529 0.266 0.523 1.18 0.83 2 1.73 1.73 1.25 1.28 1.11 1.26 1.003 0.958 0.174 0.406 0.225 2.306 1.11 1.16 3 1.82 1.84 1.32 1.37 1.51 1.50 0.985 1.004 0.470 0.468 0.359 0.339 1.35 1.32 4 2.16 2.05 1.50 1.52 1.73 1.66 1.073 1.088 0.556 0.476 0.414 0.395 1.41 1.30 5 1.92 1.89 1.34 1.36 1.48 1.57 0.974 1.010 0.482 0.492 0.370 0.371 1.35 1.35 6 1.76 1.75 1.27 1.24 1.58 1.58 0.923 0.933 0.464 0.484 0.392 0.383 1.22 1.19 7 1.82 1.82 1.31 1.31 1.58 1.59 0.961 0.973 0.440 0.407 0.396 0.366 1.14 1.19 8 1.98 1.85 1.38 1.32 1.62 1.71 0.976 0.919 0.483 0.502 0.398 0.367 1.28 1.28 9 1.73 1.75 1.26 1.26 1.50 1.51 0.919 0.943 0.460 0.462 0.355 0.361 1.28 1.12 10 1.73 1.74 1.15 1.21 1.57 1.53 0.882 0.913 0.466 0.468 0.371 0.369 1.15 1.22 Lab 4044755861 EV4H2R EV4H2Q CLC10-B 00866RF00 00859RF00 00795RF00 1 2.02 1.51 0.080 0.099 0.044 0.061 1.73 2.07 3.13 3.21 1.87 1.93 0.879 0.608 2 0.66 0.07 0.021 0.111 0.040 0.054 2.52 1.98 1.83 2.16 1.56 1.65 0.311 0.375 3 1.58 1.64 0.099 0.098 0.048 0.049 2.13 2.13 3.19 3.32 2.07 2.10 0.915 0.919 4 1.78 1.73 0.088 0.064 0.045 0.043 2.12 2.23 3.48 3.50 1.93 2.21 1.002 0.890 5 1.68 1.68 0.103 0.101 0.049 0.050 2.28 2.19 3.44 3.47 2.04 2.06 0.891 0.894 6 1.74 1.76 0.104 0.101 0.046 0.044 2.19 2.31 3.67 3.53 1.83 1.96 0.939 0.976 7 1.50 1.53 0.101 0.107 0.040 0.037 2.00 2.26 3.62 3.60 1.79 1.82 0.947 0.974 8 1.63 1.77 0.109 0.095 0.049 0.048 2.31 2.23 3.46 3.50 1.91 2.02 0.910 0.883 9 1.64 1.60 0.074 0.073 0.025 0.025 2.21 2.09 3.29 3.34 1.84 1.87 0.890 0.913 10 1.53 1.60 0.095 0.101 0.041 0.041 2.00 2.09 3.22 3.25 1.75 2.09 0.864 0.884 Lab 50350017W1 1 1.18 1.11 2 1.18 1.22 3 1.29 1.26 4 1.43 1.46 5 1.30 1.31 6 1.24 1.18 7 1.27 1.24 8 1.21 1.32 9 1.17 1.16 10 1.14 1.17 HIGHLIGHTED RED ARE RESULTS THAT WERE FLAGGED BY THE COCHRAN’S OR SINGLE/DOUBLE GRUBBS TEST AND REMOVED

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Table 6: Statistical Analysis of MLT Blind Duplicates for Riboflavin

Sample ID:

K16NTAV

E10NWZC

E10NWZC

410057652Z

410057652Z

410457651Z

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

10 20

9

10 20

9

10 20

18

18

1.8253 0.0701 0.1303

1.3044 0.1328 0.1328

1.3139 0.0267 0.0939

1.5430 0.1318 0.1702

1.5317 0.0498 0.1504

0.9595 0.0490 0.0674

Repeatability RSD, % Reproducibility RSD, %

3.84 7.14 0.24

10.18 10.18

2.03 7.15 0.23

8.54

3.25 9.82 0.33

5.11 7.02 0.22

11.03

HORRAT value

0.33

0.37

Sample ID:

00729RF00

00729RF00

00730RF00

00730RF00

4052755861

4052755861

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

9

10 20

8

10 20

9

18

16

18

0.4651 0.0588 0.0838

0.4846 0.0291 0.0458

0.4663 0.4691 0.4691 100.59 100.59

0.3754 0.0131 0.0198

1.2210 0.0940 0.1297

1.2452 0.0542 0.0943

Repeatability RSD, % Reproducibility RSD, %

12.65 18.01

6.01 9.45 0.26

3.50 5.27 0.14

7.70

4.35 7.57 0.24

10.62

HORRAT value

0.50

2.80

0.34

Sample ID:

4044755861

4044755861

EV4H2R

EV4H2R

EV4H2Q

CLC10-B

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

8

10 20

9

10 20

10 20

16

18

1.5331 0.1798 0.4370

1.6498 0.0458 0.0910

0.0911 0.0215 0.0215

0.0940 0.0081 0.0131

0.0439 0.0050 0.0086

2.1541 0.1631 0.1631

Repeatability RSD, % Reproducibility RSD, %

11.73 28.50

2.78 5.52 0.19

23.58 23.58

8.67

11.47 19.55

7.57 7.57 0.27

13.94

HORRAT value

0.95

0.51

0.31

0.38

Sample ID:

00866RF00

00866RF00

00859RF00

00795RF00

00795RF00

50350017W1

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

9

10 20

10 20

8

10 20

18

16

3.2606 0.0881 0.4751

3.4014 0.0515 0.1664

1.9150 0.1065 0.1628

0.8431 0.0685 0.1926

0.9181 0.0319 0.0397

1.2417 0.0363 0.0928

Repeatability RSD, % Reproducibility RSD, %

2.70

1.51 4.89 0.18

5.56 8.50 0.29

8.13

3.48 4.33 0.13

2.92 7.48 0.24

14.57

22.85

HORRAT value

0.54

0.70

In Red are statistics after flagged results were removed

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Total Pyridoxine MLT data are summarized in the tables below.

Table 7: AOAC 2015.14 Pyridoxine Data (µg/100g) from Analysis of Blind Duplicates, Complete Set of Individual Results Lab K16NTAV E10NWZC 410057652Z 410457651Z 00729RF00 00730RF00 4052755861 1 0.592 0.590 0.623 0.631 0.486 0.497 0.553 0.552 0.495 0.497 0.386 0.391 0.252 0.254 2 0.597 0.627 0.595 0.608 0.636 0.684 0.552 0.528 1.082 1.210 0.950 3.254 0.355 0.340 3 0.659 0.647 0.592 0.585 0.506 0.527 0.520 0.514 0.445 0.441 0.353 0.338 0.463 0.434 4 0.703 0.653 0.602 0.643 0.514 0.525 0.549 0.555 0.473 0.473 0.373 0.365 0.383 0.367 5 0.656 0.670 0.610 0.616 0.524 0.525 0.533 0.541 0.482 0.477 0.371 0.372 0.435 0.432 6 0.719 0.705 0.633 0.628 0.560 0.546 0.562 0.544 0.493 0.499 0.395 0.370 0.487 0.474 7 0.603 0.606 0.656 0.658 0.523 0.537 0.500 0.501 0.472 0.468 0.373 0.376 0.363 0.364 8 0.621 0.595 0.571 0.571 0.506 0.525 0.528 0.503 0.471 0.447 0.376 0.353 0.421 0.432 9 0.659 0.662 0.569 0.578 0.554 0.557 0.539 0.545 0.515 0.495 0.383 0.387 0.449 0.412 10 0.690 0.680 0.608 0.590 0.514 0.515 0.548 0.551 0.499 0.483 0.382 0.366 0.458 0.422 Lab 4044755861 EV4H2R EV4H2Q CLC10-B 00866RF00 00859RF00 00795RF00 1 0.644 0.634 0.056 0.055 0.002 0.002 1.35 1.37 3.11 3.07 1.94 1.94 0.504 0.502 2 0.844 0.090 0.051 0.045 0.022 0.027 1.48 1.54 3.40 3.55 1.98 1.88 0.542 0.529 3 0.607 0.624 0.050 0.051 0.003 0.003 1.45 1.32 2.87 2.85 1.86 1.86 0.451 0.442 4 0.667 0.649 0.054 0.056 0.000 0.000 1.42 1.36 3.12 3.01 1.93 1.94 0.494 0.490 5 0.668 0.674 0.054 0.054 0.002 0.002 1.43 1.41 3.08 3.05 1.97 1.96 0.486 0.490 6 0.676 0.699 0.053 0.054 0.001 0.001 1.55 1.45 3.16 3.08 1.99 1.98 0.511 0.481 7 0.661 0.658 0.052 0.052 0.004 0.003 1.33 1.39 2.98 3.04 1.72 1.73 0.451 0.443 8 0.607 0.626 0.053 0.053 0.001 0.001 1.40 1.35 2.98 2.98 1.86 1.82 0.457 0.465 9 0.679 0.696 0.057 0.055 0.002 0.002 1.41 1.43 3.28 3.11 1.89 1.84 0.566 0.588 10 0.662 0.656 0.052 0.055 0.002 0.002 1.48 1.37 2.99 3.10 1.95 2.16 0.476 0.496 Lab 50350017W1 1 0.313 0.305 2 0.405 0.347 3 0.355 0.367 4 0.366 0.355 5 0.373 0.376 6 0.401 0.401 7 0.325 0.333 8 0.357 0.348 9 0.357 0.345 10 0.368 0.360 HIGHLIGHTED RED ARE RESULTS THAT WERE FLAGGED BY THE COCHRAN’S OR SINGLE/DOUBLE GRUBBS TEST AND REMOVED

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Table 8: Statistical Analysis of MLT Blind Duplicates for Total Pyridoxine

Sample ID:

K16NTAV

E10NWZC

410057652Z 410457651Z

00729RF00

00729RF00

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

10 20

10 20

10 20

10 20

9

18

0.6467 0.0153 0.0420

0.6083 0.0111 0.0282

0.5380 0.0136 0.0477

0.5358 0.0093 0.0196

0.5459 0.0298 0.2127

0.4792 0.0086 0.0209

Repeatability RSD, % Reproducibility RSD, %

2.37 6.50 0.19

1.82 4.63 0.13

2.53 8.86 0.25

1.73 3.66 0.10

5.45

1.80 4.37 0.12

38.97

HORRAT value

1.11

Sample ID:

00730RF00

00730RF00 4052755861 4044755861 4044755861

EV4H2R

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

9

10 20

10 20

9

10 20

18

18

0.5457 0.5152 0.6572

0.3727 0.0100 0.0146

0.3998 0.0146 0.0671

0.6361 0.1688 0.1688

0.6549 0.0104 0.0277

0.0531 0.0016 0.0026

Repeatability RSD, % Reproducibility RSD, %

94.42

2.68 3.93 0.11

3.66

26.55 26.55

1.59 4.24 0.12

2.95 4.95 0.10

120.45

16.77

HORRAT value

3.44

0.46

0.78

Sample ID:

EV4H2Q

CLC10-B

00866RF00

00859RF00

00795RF00

50350017W1

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

10 20

10 20

10 20

10 20

10 20

0.0040 0.0013 0.0073

1.4140 0.0513 0.0646

3.0907 0.0674 0.1695

1.9089 0.0551 0.0993

0.4932 0.0106 0.0407

0.3579 0.0142 0.0274

Repeatability RSD, % Reproducibility RSD, %

32.63

3.63 4.57 0.15

2.18 5.48 0.20

2.89 5.20 0.18

2.14 8.25 0.23

3.97 7.65 0.20

184.23

HORRAT value

2.51

In Red are statistics after flagged results were removed

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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Total Niacin MLT data are summarized in the tables below.

Table 9: AOAC 2015.14 Total Niacin Data (µg/100g) from Analysis of Blind Duplicates, Complete Set of Individual Results Lab K16NTAV E10NWZC 410057652Z 410457651Z 00729RF00 00730RF00 4052755861 1 8.94 9.37 10.7 10.5 6.19 6.64 8.98 8.97 4.30 4.26 3.023 3.287 4.87 4.85 2 7.34 6.58 6.83 6.28 5.30 6.64 6.60 5.96 6.23 6.80 5.435 17.312 4.04 3.77 3 9.05 9.13 9.53 9.89 7.36 7.70 7.83 8.56 4.38 4.33 3.087 3.346 5.28 5.06 4 8.74 8.50 8.63 8.68 6.41 6.42 7.46 7.39 3.97 3.93 2.911 2.942 4.42 4.43 5 8.90 8.89 8.45 8.48 6.44 6.43 7.48 7.35 4.19 4.20 3.146 3.395 4.77 4.54 6 8.33 8.22 8.21 7.87 6.25 6.36 6.97 6.81 4.28 4.23 2.969 2.978 4.36 4.42 7 8.14 8.15 8.35 8.34 7.05 6.95 7.06 7.04 4.50 4.48 3.041 2.996 4.53 4.58 8 8.35 8.81 7.95 8.24 6.06 6.24 7.22 7.03 3.86 3.74 3.182 2.931 4.65 4.57 9 8.19 7.97 8.83 8.83 6.87 6.62 7.41 7.38 4.09 3.95 3.087 3.034 4.26 4.05 10 8.14 8.57 8.41 8.87 7.44 6.81 7.00 7.43 4.14 3.90 2.94 3.01 4.58 4.99 Lab 4044755861 EV4H2R EV4H2Q CLC10-B 00866RF00 00859RF00 00795RF00 1 6.41 6.37 0.945 0.929 0.00 0.00 11.1 11.3 11.0 10.9 15.1 14.7 13.5 13.7 2 6.36 0.74 0.627 5.628 0.03 0.03 10.3 10.9 9.4 9.7 14.2 14.5 9.9 9.7 3 7.56 7.11 0.976 0.986 0.02 0.02 10.3 11.2 11.7 12.2 16.5 17.3 14.2 13.9 4 5.76 6.09 0.910 0.917 0.00 0.00 10.1 10.4 10.8 10.6 14.3 14.4 13.9 13.6 5 6.03 6.34 0.957 0.970 0.01 0.01 10.8 10.8 10.9 10.5 13.4 13.5 13.1 13.4 6 6.13 6.16 0.925 0.929 0.00 0.00 10.1 10.8 10.8 11.0 14.0 14.0 12.6 12.8 7 6.83 6.71 0.960 0.999 0.03 0.03 10.5 10.4 11.6 11.6 15.2 15.2 13.2 13.1 8 5.93 6.26 0.862 0.856 0.02 0.02 9.9 10.4 10.3 10.0 14.6 14.5 13.0 13.2 9 5.03 5.46 0.851 0.882 0.01 0.01 9.7 9.8 9.0 8.7 14.3 14.3 12.5 12.8 10 6.05 6.32 0.936 0.926 0.01 0.01 10.4 11.8 11.5 10.8 14.5 17.3 12.8 13.3 Lab 50350017W1 1 4.67 4.72 2 3.91 4.11 3 4.53 4.64 4 4.21 4.22 5 4.38 4.33 6 4.07 4.09 7 4.06 4.03 8 4.16 4.30 9 3.99 3.86 10 4.54 4.47 HIGHLIGHTED RED ARE RESULTS THAT WERE FLAGGED BY THE COCHRAN’S OR SINGLE/DOUBLE GRUBBS TEST AND REMOVED

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Table 10: Statistical Analysis of MLT Blind Duplicates for Total Niacin

Sample ID:

K16NTAV

E10NWZC

410057652Z 410457651Z

00729RF00

00729RF00

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

10 20

10 20

10 20

10 20

9

18

8.4145 0.2533 0.6589

8.5906 0.2120 1.0647

6.6083 0.3621 0.5474

7.3954 0.2458 0.7552

4.3843 0.1455 0.7816

4.1480 0.0743 0.2239

Repeatability RSD, % Reproducibility RSD, %

3.01 7.83 0.34

2.47

5.48 8.28 0.34

3.32

3.32

1.79 5.40 0.21

12.39

10.21

17.83

HORRAT value

0.54

0.43

0.70

Sample ID:

00730RF00

00730RF00 4052755861 4044755861 4044755861

EV4H2R

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

9

10 20

10 20

9

10 20

18

18

3.9022 2.6583 3.2307

3.0720 0.1230 0.1467

4.5507 0.1412 0.3739

5.9816 1.2742 1.3543

6.2518 0.2090 0.5921

1.1486 1.1183 1.1183

Repeatability RSD, % Reproducibility RSD, %

68.12 82.79

4.01 4.78 0.18

3.10 8.22 0.32

21.30 22.64

3.34 9.47 0.39

97.36 97.36

HORRAT value

3.18

0.93

3.11

Sample ID:

EV4H2R

EV4H2Q

CLC10-B

00866RF00

00859RF00

00795RF00

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

9

10 20

10 20

10 20

10 20

10 20

18

0.9287 0.0133 0.0452

0.0135 0.0009 0.0115

10.5432 0.4446 0.5295

10.6455 0.2681 0.9505

14.8004 0.6479 1.0982

12.9252 0.1837 1.1846

Repeatability RSD, % Reproducibility RSD, %

1.44 4.86 0.15

6.66

4.22 5.02 0.22

2.52 8.93 0.40

4.38 7.42 0.35

1.42 9.16 0.42

85.04

HORRAT value

1.39

Sample ID:

50350017W1

Total number of laboratories Total number of replicates Overall mean of all data, µg/100g Repeatability standard deviation Reproducibility standard deviation

10 20

4.2641 0.0698 0.2665

Repeatability RSD, % Reproducibility RSD, %

1.64 6.25 0.24

HORRAT value

In Red are statistics after flagged results were removed

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Repeatability (RSD r ) Thiamine

For all fourteen of the fortified product matrices analyzed, the overall repeatability averaged 2.5% RSD with a range of 1.39-4.4% RSD and all met the standard method performance requirements of ≤ 5% RSD. The Thiamine level in CLC10-B averaged 1.37 µg/100g. Riboflavin For all fourteen of the fortified product matrices analyzed, the overall repeatability averaged 5.0% RSD with a range of 1.51-8.67% RSD and 10 of the 14 met the standard method performance requirements of ≤ 5% RSD. The Riboflavin level in CLC10-B averaged 2.15 µg/100g. Total Pyridoxine For all fourteen of the fortified product matrices analyzed, the overall repeatability averaged 2.4% RSD with a range of 1.59-3.97% RSD and all met the standard method performance requirements of ≤ 5% RSD. The total Pyridoxine level in CLC10-B averaged 1.41 µg/100g. Total Niacin For all fourteen of the fortified product matrices analyzed, the overall repeatability averaged 2.5% RSD with a range of 1.42-5.48% RSD and all met the standard method performance requirements of ≤ 5% RSD. The total Niacin level in CLC10-B averaged 10.5 µg/100g.

Reproducibility (RSD R ) Thiamine

For all product matrices, reproducibility averaged 10.8% RSD with a range of 6.01-23.4% RSD. Nine of the 14 fortified products met the standard method performance requirements for reproducibility of ≤ 10% RSD for fortified products. I will discuss this in the general comments section. Riboflavin For all product matrices, reproducibility averaged 7.8% RSD with a range of 4.33-13.9% RSD. Thirteen of the 14 fortified products met the standard method performance requirements for reproducibility of ≤ 10% RSD for fortified products. Total Pyridoxine

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For all product matrices, reproducibility averaged 6.6% RSD with a range of 3.66-16.8% RSD. Thirteen of the 14 fortified products met the standard method performance requirements for reproducibility of ≤ 10% RSD for fortified products. Total Niacin For all product matrices, reproducibility averaged 7.9% RSD with a range of 4.78-12.4% RSD. Thirteen of the 14 fortified products met the standard method performance requirements for reproducibility of ≤ 10% RSD for fortified products. Statistical Analysis of MLT Data Using the AOAC International Interlaboratory Study Workbook for Blind (unpaired) Replicates, Version 2.1 to analyze B 1 , B 2 , B 3 , and B 6 MLT data, statistical outliers were identified. There was a total of 21 lab/sample/analyte pairs of data that were removed when flagged. 17 of these came from lab 2 as one of their days of data had many anomalies. I did reach out to see if they could evaluate the data after finding contact information but have not received a response as of the date of this report. Lab 1 had significant deviations in Riboflavin standards. These deviations carried through to the samples and 6 of their 15 sample/analyte pairs were flagged and removed. There were 3 samples where both lab 1 and lab 2 were removed, resulting in 8 labs of data. Outside of Riboflavin for lab 1, and sporadic data from lab 2, no other statistical flags resulted in removal of data. General Comments In general, B vitamins are stable, but Thiamine is slightly sensitive to temperature, oxygen, humidity, and light. This study has taken over two years and there does seem to be a downward trend of thiamine data during this time. This may help explain why repeatability did not fail in any of the edited sample sets, but reproducibility was above the allowed 10% for 5 of the 14 fortified samples. There were several deviations to the MLT protocol during the MLT study, but none of these deviations appeared to impact final results. One laboratory did not analyze the practice samples. One laboratory used a sample size outside of the recommended weight. There were at least 2 variations of enzyme cocktail that were used.

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Appendix A:

Method

Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3 by Enzymatic Digestion and LC-MS/MS. Louis M. Salvati, Sean C. McClure, and Nicholas A. Cellar. Abbott Nutrition, 3300 Stelzer Rd. Columbus, OH 43219

, and B 6

in Infant Formula and Related Nutritionals

Introduction AOAC SPIFAN released a call for methods for total vitamins B 1 , B 2 , B 3 pyrophosphate, and thiamine triphosphate in the SMPR. 1 Total B 2 phosphate, and flavin adenine dinucleotide. 2 Total B 3 nutritionals. Total vitamin B 1

, and B 6

in infant formula and related

is described as the sum of thiamine, thiamine monophosphate, thiamine

is defined as riboflavin, riboflavin-5’-

is defined as the sum of nicotinic acid and

nicotinamide. 3 Finally, total B 6 includes five forms: pyridoxine, pyridoxal, pyridoxal-5’-phosphate, pyridoxamine, and pyridoxamine-5’-phosphate. 4 A single method for all four total vitamins was developed to meet the need.

The combined method was developed to measure thiamine, riboflavin, pyridoxamine, pyridoxal, pyridoxamine, nicotinic acid, and nicotinamide directly. Separation was achieved with 20 mM ammonium formate mobile phase without ion pairing agent. Improved retention of thiamine has been previously demonstrated by increasing the mobile phase pH. 5 In fact, there is a striking improvement in water soluble vitamin retention under reverse phase conditions at moderate pH (5 -7). 6 Despite this improvement in retention, elution of phosphorylated compounds is notoriously difficult. 7 The phosphate moiety complexes with Fe 3+ and thus phosphate containing compounds tend to tail considerably with conventional chromatographic equipment due to stainless steel plumbing and column frits. 7 Further, the phosphorylated forms of these vitamins tend to be a small fraction of the free-form thus putting pressure on accuracy of quantitation. Instead of trying to overcome the challenges of direct analysis of the phosphorylated forms, this method includes an overnight, enzymatic digestion with acid phosphatase to hydrolyze the phosphate yielding the free vitamin forms. 5 Papain and α-amylase are also included in the enzymatic cocktail to digest protein and complex carbohydrate, respectively, thus aiding reduction in sample extract complexity. 8 Method performance was demonstrated in 14 SPIFAN II matrixes. Precision averaged 3.9%, and over-spike recovery was generally 95-105%. Both free and phosphorylated forms were over-spiked as part of the recovery experiment. The method was demonstrated to meet both SPIFAN SMPR requirements and Abbott Nutrition requirements.

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AOAC Official Method 2015.14 Simultaneous Determination of Total Vitamins B 1 , B 2 , B 3

, and B 6

, in Infant Formula and Related Nutritionals

by Enzymatic Digestion and LC-MS/MS. First Action 2015

A.

Principle This method facilitates simultaneous quantitation of four water-soluble vitamins (WSV) in infant formula and related nutritional products, including all SPIFAN SMPR relevant forms of vitamins B 1 , B 2 , B 3 , and B 6 . Samples are prepared by enzymatic digestion with papain and α-amylase to hydrolyze protein and complex carbohydrate and acid phosphatase to free phosphorylated vitamin forms. Stable-isotope labeled internal standards are incorporated into the sample preparation to correct for variability in both the sample preparation and instrument response. A series of six mixed working standard solutions spanning two orders of magnitude in vitamin concentration are used to generate calibration curves based on the peak response ratio of the analyte to its stable-isotope labeled internal standard. Prepared samples and working standard solutions are injected onto ultra-high pressure liquid chromatograph (UPLC) interfaced to a triple-quadrupole mass spectrometer (MS/MS) for analysis. The MS/MS is configured to monitor parent-daughter (precursor-fragment) ion pairs for each analyte and internal standard. This reaction forms the basis for method selectivity. Analytes are quantified by least squares regression using the response ratio of the analyte to its internal standard. B. Apparatus and Materials 1. Control Sample: NIST SRM 1849a, or current lot. Store at 4°C. 2. Waters Acquity BEH C18 column, 2.1 x 100, 1.7 μm. Part# 186002352. 3. UPLC system: Waters Acquity Classic, or equivalent. 4. Tandem quadrupole mass spectrometer with ESI probe: Waters Xevo TQ-S, or equivalent. 5. Analytical balances (3): a) One capable of accurately weighing 5.00 mg (for standards), 6-place balance. b) An analytical five-place balance for samples, and c) A top loading two place balance capable of weighing to several hundred grams.

6. Water purifier: Millipore Milli-Q Water Purification System, or equivalent. 7. Water bath shaker capable of maintaining 37°C, Lab-Line Orbit, or equivalent. 8. Bottle-top dispenser: capable of dispensing volumes of approximately 24 mL. 9. pH meter: capable of measuring a pH of 4.0 - 5.0. 10.Vortex mixer.

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11.Multi-position magnetic stir plate. 12.Room Light Shields: A.L.P. Protect-A-Lamp, UV cutoff at 460 nm, or equivalent. 13.Graduated cylinders: various sizes, including: 10, 100, 500, and 1000 mL. 14.Beakers: various sizes, including: 100, 200, 400, 600, 1000, and 2000 mL. 15.Volumetric flasks: various sizes, including: 10, 25, 50, 100, 250, and 2000 mL. 16.Mobile phase bottles: glass, various sizes, including: 250, 500, 1000, and 2000 mL . 17.Disposable, plastic Pasteur pipettes. 18.Amber bottles: volume capacity of 50 and 100 mL (for stock standard storage). 19.Weighing vessels: various, including disposable weighing boats and glass weighing funnels. 20.Positive displacement pipettes: Gilson Microman - 10, 100, 250, and 1000 µL; Part #’s F148501, F148504, F148505, and F148506. 21.Positive displacement pipette tips: Gilson Capillary Piston - 10, 100, 250, and 1000 µL; Part #’s F148312, F148314, F148014, and F148560. 22.Plastic syringes, 3 mL. 23.Syringe filters, PTFE 0.45 µm syringe filters: Acrodisc 25 mm, or equivalent. 24.50 mL self-standing, plastic centrifuge tubes: Superior Scientific, Ltd., or equivalent. 25.Autosampler vials: Waters autosampler vials; 9 mm amber with screw top 12x32 mm pre-split PTFE- silicon septa; Waters Part # 186000847C, or equivalent. 26.Teflon coated magnetic stir bars. C. Reagents 1. Nicotinamide: USP Reference Standard (U.S.P.C., Inc. Rockville, MD) Official Lot; Catalog # 1462006. Store as indicated on label. 2. Niacin (nicotinic acid): USP Reference Standard (U.S.P.C., Inc. Rockville, MD) Official Lot; Catalog # 1461003. Stored as indicated on label. 3. Pyridoxine hydrochloride: USP Reference Standard (U.S.P.C., Inc. Rockville, MD) Official Lot; Catalog # 1587001. Store in desiccator protected fromwhite light. Dry according to manufacturer’s instructions prior to use. 4. Riboflavin: USP Reference Standard (U.S.P.C., Inc. Rockville, MD) Official Lot; Catalog # 1603006. Store in desiccator protected from white light. Dry according to manufacturer’s instructions prior to use. 5. Thiamine hydrochloride: USP Reference Standard (U.S.P.C., Inc. Rockville, MD) Official Lot; Catalog # 1656002. Store in desiccator protected fromwhite light. Measure the moisture content of the powder prior to use. 6. Pyridoxamine dihydrochloride: Fluka Analytical Standard, catalog# P9380. 7. Pyridoxal hydrochloride: Sigma, catalog# P9130. 8. 2 H 4 -Nicotinamide: CDN Isotopes; Catalog # D-3457. 9. 2 H 4 -Nicotinic acid: CDN Isotopes; Catalog # D-4368.

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10. 13 C 4

-Pyridoxine: pyridoxine:HCl (4,5-bis(hydroxymethyl)- 13 C 4

); Cambridge Isotope Laboratory;

Catalog # CLM-7563.

11. 2 H 3 12. 2 H 3 13. 13 C 4 14. 13 C 4

-Pyridoxal: IsoSciences; Catalog # 7098. -Pyridoxamine: IsoSciences; Catalog # 7099. -Thiamine chloride; IsoSciences; Catalog # 9209.

, 15 N 2 -Riboflavin: IsoSciences, Catalog# 7072. 15.Acid phosphatase, type II from potato, 0.5 – 3.0 U/mg: Sigma, catalog# P3752. 16.Papain from Carica papaya, ≥ 3 U/mg: Sigma, catalog# 76220. 17.α–amylase from aspergillus oryzae, 150 U/mg: Sigma, catalog# A9857. 18.Hydrochloric acid concentrated (12M); ACS grade, or equivalent. 19.Ammonium formate: for mass spectrometry (≥99.0%), Fluka 70221 or equivalent.

20.Glacial acetic acid: Sigma ACS Reagent Grade, or equivalent. 21.Formic acid: Sigma ACS Reagent Grade, or equivalent. 22.Laboratory Water: 18.0 MΩ, < 10 ppb TOC, or equivalent. 23.Methanol: Fisher LC-MS/MS Optima grade or EMD Omni-Solve LC-MS grade. 24.Ethylenediaminetetracetic acid, disodium salt dihydrate (EDTA): ACS grade (99-101%), or equivalent.

25.Potassium phosphate dibasic: ACS grade (>98%), or equivalent. 26.meta-Phosphoric acid: ACS grade (33.5-36.5%), or equivalent. 27.pH 4.0, 7.0, and 10.0 buffer solutions for pH meter calibration. 28.Phosphoric acid, 85%, ACS grade, or equivalent. 29.Potassium hydroxide, 40%, ACS grade, or equivalent.

D.

Standard and Solution Preparation 1. Mobile Phase A: 20 mM ammonium formate in water - Using a graduated cylinder, transfer 500 mL laboratory water to a mobile phase reservoir. Add 0.631 g of ammonium formate and mix well. Scale as needed. Expiration: 3 days. 2. Mobile Phase B: Methanol. 3. Weak needle wash: 10% methanol in water, expiration 3 mo. 4. Strong needle wash: Methanol. 5. 50 mM ammonium formate - Using a graduated cylinder, transfer 1400 mL laboratory water to an appropriate reservoir. Add 4.41 g of ammonium formate and mix well. 1400 mL is adequate for 6 working standards and 32 samples. Scale as needed. Expiration: 3 days. 6. Enzyme Cocktail - Using a graduated cylinder, transfer 200 mL 50 mM ammonium formate buffer to an appropriate reservoir. Add 200 ± 10 mg acid phosphatase and 200 ± 10 mg papain. Mix for ten

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minutes with magnetic stir plate and stir bar. Adjust to pH 4.0-4.5 with formic acid (~ 100 μL). 200 mL is adequate for 6 working standards and 32 samples. Scale as needed. Prepare fresh daily. 5. Stable-Isotope Labeled Compounds – Individual, Internal Standard Stock Solutions a) 2 H 4 -Nicotinamide Stock Solution (Approximate Concentration: 560 µ g/mL) - Weigh 14.0 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 50 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. b) 2 H 4 -Nicotinic Acid Stock Solution (Approximate Concentration: 500 µ g/mL) - Weigh 12.5 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 25 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 50 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. c) 13 C 4 -Pyridoxine Stock Solution (Approximate Concentration: 70 µ g/mL) - Weigh 7.0 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. d) 2 H 3 -Pyridoxal Stock Solution (Approximate Concentration: 40 µ g/mL) - Weigh 4.0 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. e) 2 H 3 -Pyridoxamine Stock Solution (Approximate Concentration: 40 µ g/mL) - Weigh 4.0 ± 0.1 mg into a tarred weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with laboratory water and QS with laboratory water. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. e) 13 C 4 -Thiamine Chloride Stock Solution (Approximate Concentration: 100 µ g/mL) i) 0.12 M HCl - Add approximately 300 mL water to a 500 mL graduated cylinder. Add 5.0 ± 0.1 mL conc. HCl and swirl to mix. Bring to 500 mL with laboratory water and mix well. ii) Weigh 5.0 ± 0.1 mg 13 C 4 -thiamine into a tarred weighing vessel. Quantitatively transfer to a 50 mL volumetric flask with 0.12 N HCl and QS with 0.12 N HCl. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. f) 13 C 4 , 15 N 2 -Riboflavin Stock Solution (Approximate Concentration: 73 µ g/mL) i) 1.0% Acetic acid in water - Add approximately 30 mL water to a 500 mL graduate cylinder. Add 5.0 ± 0.1 mL glacial acetic acid and swirl to mix. Bring to 500 mL with laboratory water and mix well. ii) Weigh 7.3 ± 0.1 mg 13 C 4 , 15 N 2 -riboflavin into a tarred weighing vessel. Quantitatively transfer to a 100 mL volumetric flask with 1.0% acetic acid and QS with 1.0% acetic acid. Mix well and transfer to a 100 mL amber bottle and Store refrigerated (2˚-8˚C). Expiration: Until exhausted or evidence of contamination. 6. Internal Standard Stock Mixture (ISSM) – Combine 2500 μL of 50 mM ammonium formate with 250 μL 2 H 4 -nicotinamide stock, 250 μL 2 H 4 -nicotinic acid stock, 250 μL 13 C 4 -pyridoxine stock, 200 μL 2 H 3 - pyridoxal stock, 50 μL 2 H 3 -pyridoxamine stock, 250 μL 13 C 4 -thiamine stock, and 250 μL 13 C 4 , 15 N 2 -

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

FOR ERP USE ONLY DO NOT DISTRIBUTE riboflavin acid stock. Volume provides sufficient ISSM for 6 working standards and 32 samples. Scale as needed. Prepare fresh daily. 7. Phosphate Buffer Solution, pH 5.0 (0.10M Potassium Phosphate Dibasic, 1% EDTA, 2% Metaphosphoric Acid) . a) Weigh 20.0 ± 0.2 g of EDTA into a tarred weighing vessel and quantitatively transfer to a 2000 mL beaker containing approximately 1800 mL laboratory water and add a magnetic stir bar. b) Weigh 34.8 ± 0.1 g of potassium phosphate dibasic into a tarred weighing vessel and quantitatively transfer to the 2000 mL beaker already containing approximately 1800 mL laboratory water and EDTA. Mix by stirring on a magnetic stir plate until both the EDTA and potassium phosphate dibasic is completely dissolved. c) Weigh 40.0 ± 0.2 g grams of metaphosphoric acid into a tarred weighing vessel and quantitatively transfer to the 2000 mL beaker containing approximately 1800 mL laboratory water, EDTA, and potassium phosphate dibasic. Mix by stirring on a magnetic stir plate until the metaphosphoric acid is completely dissolved. Adjust the pH of the solution to pH 5.00 ± 0.02 using 40% potassium hydroxide or 85% phosphoric acid. Quantitatively transfer the solution to a 2000 mL volumetric flask and dilute to volume with laboratory water. Expiration: 48 hours. 8. Native Compounds – Stock Standard Solutions a) Vitamin Standard Stock Mixture (VSSM) - Accurately weigh the indicated amounts for the following standards using separate weighing funnels or other appropriate weighing vessel and quantitatively transfer to a 100 mL volumetric flask using phosphate buffer (pH 5): (a) Niacinamide: 70.5 ± 0.5 mg (b) Thiamine hydrochloride: 10.5 ± 0.2 mg Determine the moisture of the USP thiamine hydrochloride reference standard as directed on the container immediately prior to weighing. The percent moisture determined for the reference standard is used to calculate the concentration of thiamine in the VSSM. (c) Riboflavin: 7.0 ± 0.2 mg - Dry an appropriate amount of the USP riboflavin reference standard at 105 (±1) °C for 2 hours (± 10 minutes) prior to weighing. (d) Pyridoxine Hydrochloride: 10.8 ± 0.2 mg - Dry an appropriate amount of the USP pyridoxine hydrochloride reference standard over indicating absorbent in vacuo for 4 hours prior to weighing. QS to volume with phosphate buffer (pH 5) solution. Heat and slowly stir until the standards have completely dissolved (riboflavin dissolves more slowly) and the solution is clear. Do not heat the solution for more than 40 minutes and do not exceed 90°C. Store refrigerated (2˚-8˚C). Expiration: 3 months. b) Nicotinic Acid Stock Solution (550 ug/mL) - Accurately weigh 13.7 ± 0.1 mg USP niacin (nicotinic acid, catalog 1461003) reference standard. Quantitatively transfer the nicotinic acid to a 25 mL volumetric flask. Add lab water to a total volume of about 20 mL and swirl until completely dissolved. Bring to volume with lab water. Mix well. Expiration: 3 months.

2015.14 (JULY 2018) BVit-02 Total B_AOAC_2015.14_MLT_Final

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c) Pyridoxal Stock Solution (140 ug/mL) - Accurately weigh 17.0 ± 0.5 mg pyridoxal dihydrochloride standard. Quantitatively transfer to a 100 mL volumetric flask. Add lab water to a total volume of about 70 mL and swirl until completely dissolved. Bring to volume with lab water. Mix well. Expiration: 3 months. d) Pyridoxamine Stock Solution (160 ug/mL) - Accurately weigh 23.0 ± 0.5 mg pyridoxamine hydrochloride standard. Quantitatively transfer to a 100 mL volumetric flask. Add lab water to a total volume of about 70 mL and swirl until completely dissolved. Bring to volume with lab water. Mix well. Expiration: 3 months. 9. Mixed Working Standard (MWS) - Combine 500 μL VSSM, 25 μL pyridoxamine stock, 25 μL pyridoxal stock, and 65 μL nicotinic acid stock solutions in a 10 mL volumetric flask containing approximately 5 mL of 50 mM ammonium formate. Bring to volume with 50 mM ammonium formate and mix well. Prepare fresh daily. 10. Working Standard Solution Preparation a) WS1 – Add 20 μL MWS and 980 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. b) WS2 – Add 50 μL MWS and 950 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. c) WS3 – Add 100 μL MWS and 900 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. d) WS4 – Add 200 μL MWS and 800 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. e) WS5 – Add 500 μL MWS and 500 μL of 50 mM ammonium formate to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. f) WS6 – Add 1000 μL MWS to a 50 mL centrifuge tube. Add 100 μL of ISSM, and vortex to mix. Prepare fresh daily. E. Procedure 1. Sample preparation a) For powdered products, using a tarred beaker or LDPE cup, weigh 10.0 ± 0.3 g of sample. Record the weight to at least 4 significant figures. This is the powder weight. Add room temperature laboratory water to bring the total reconstituted sample weight (to include the product weight) to 100 ± 2 g. Record the weight to at least 4 significant figures. This is the reconstitution weight. Carefully add a stir bar so as not to splash the liquid from the beaker/cup and place it onto a stir plate. Set the stir plate to stir the sample as fast as possible without causing the sample to splatter or froth. Powder samples should stir for at least 10 minutes but not more than 30 minutes. b) For reconstituted powders and liquid products - using a tarred, 50 mL centrifuge tube, weigh the appropriate sample amount (1.000 ± 0.100 g for infant formula, 0.500 ± 0.050 g for pediatric formulas and the NIST SRM, and 0.250 ± 0.050 g for adult nutritionals). Record the weight to 0.0001 g. This is the sample weight. Add 100 µL of the internal standard mixture (ISSM) via positive-displacement pipette. Vortex to mix.