AOAC SPIFAN Review Team Meeting Book-July 22, 2015

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

REVIEW TEAM MEETING

Wednesday, July 22, 2015

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

UNITED STATES dboyd@aoac.org 301.924.7077 x126

AOAC INTERNATIONAL

SPIFAN II Review Team (Carotenoids & B Vitamins) Wednesday, July 22, 2015

Table of Contents

Meeting Agenda .....................................................................................................................................5

List of Submitted Methods .....................................................................................................................6

Carotenoids..........................................................................................................................................

 Carotenoids SMPR .......................................................................................................................9

 Carot-01 ....................................................................................................................................11

B Vitamins ...........................................................................................................................................

 B Vitamins SMPRs......................................................................................................................17

 BVit-01 .......................................................................................................................................21

 BVit-02 ......................................................................................................................................82

AOAC INTERNATIONAL Stakeholder Panel for Infant Formula and Adult Nutritionals (SPIFAN) AOAC SPIFAN REVIEW MEETING DRAFT AGENDA (6 th Set of Priority Nutrients/B Vitamins & Carotenoids)

Wednesday, July 22, 2015 1:00PM ET

1) WELCOME & INTRODUCTIONS (Sullivan)

2) DISCUSS METHOD(S) SUBMITTED

a) Carotenoids

b) B Vitamins

i)

Vitamin B 1

ii) Vitamin B 2

iii) Vitamin B 3

iv) Vitamin B 6

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 INTERNATIONAL Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN) Call for Methods Submission for B Vitamins and Carotenoids (July 15, 2015)

Designation

First Name

Last Name

Organization

E-mail Address

Nutrient

Method Title

Method Applicability

Comments:

Infant Formula and Adult/Pediatric Nutritional Formula

Carot-01

Martijn

Vermeulen

TNO Triskelion

Martijn.Vermeulen@TNO.Triskelion.nl

Carotenoids Determination of Carotenoids in Infant Formula and Adult/Pediatric Nutritional Formula using High-Performance Liquid Chromatography with Photo Diode Array Detection.

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

Multi-nutrient method for B-vitamins: Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and

VitB-01

Saovaros

Srichimuttayomphol Mead Johnson Nutrition saovaros.srichaimuttayompol@mjn.com

Vitamin B6 Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and

Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

Infant and Adult Nutritional beverages.

Method covers all 4 B Vitamins

VitB-02

Louis

Salvati

Abbott Laboratories

louis.salvati@abbott.com

Vitamin B6 Simultaneous Determination of Total Vitamin B6, B2, B3 and B1 in Infant Formula Products by LC- MS/MS Using Enzymatic Digestion

AOAC SMPR 2014.014

Standard Method Performance Requirements for Determination of Selected Carotenoids in Infant and Adult/Pediatric Nutritional Formula

Figure 3. Molecular structure of all- trans lutein.

Intended Use: Reference Method for Dispute Resolution

1 Applicability Determinations of all- trans α-carotene (CAS 7488-99-5), cis isomers of α-carotene, all- trans β-carotene (CAS 7235-40-7), cis isomers of β-carotene, all -trans lutein (CAS 127-40-2), cis isomers of lutein, and lycopene 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 Accuracy (corresponds to the VIM definition for “trueness”). — The closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. 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. α-Carotene .—All- trans α-carotene (IUPAC name: 1,3,3-trimethyl-2-[(1E,3E,5E,7E,9E,11E,13E,15E,17E)- 3,7,12,16-tetramethyl-18-(2,6,6-trimethylcyclohex-2-en-1-yl) octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]cyclohexene, CAS No.: 7488-99-5) and its cis isomers (Figure 1). β-Carotene .—All- trans ß-carotene (IUPAC name: 1,3,3-trimethyl-2-[(1E,3E,5E,7E,9E,11E,13E,15E,17E)- 3,7,12,16-tetramethyl-18-(2,6,6-trimethylcyclohexen-1-yl)

Figure 4. Molecular structure of lycopene.

octadeca-1,3,5,7,9,11,13,15,17-nonaenyl]cyclohexene, CAS No.: 7235-40-7) and its cis isomers (Figure 2). Carotenoids .—A class of organic pigments consisting of four 10-carbon terpene units, which in turn are formed from eight 5-carbon isoprene units. For the purposes of this standard, only the four carotenoids listed in the Applicability section are considered biologically important enough for consideration in this standard for in infant formula/adult nutritionals. 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. Lutein .— All -trans lutein (IUPAC name: (1R)-4-[(1E,3E,5E,7E, 9E,11E,13E,15E,17E)-18-[(1R,4R)-4-hydroxy-2,6,6-trimethyl- c y c l oh e x - 2 - e n - 1 - y l ] - 3 , 7 , 12 , 16 - t e t r ame t hy l o c t a d e c a - 1,3,5,7,9,11,13,15,17-nonaenyl]-3,5,5-trimethylcyclohex-3-en-1- ol, CAS No.: 127-40-2) and its cis isomers (Figure 3). Lycopene .—IUPAC name: (6E,8E,10E,12E,14E,16E,18E, 20E,22E,24E,26E)-2,6,10,14,19,23,27,31-octamethyldotriaconta- 2,6,8,10,12,14,16,18,20,22,24,26,30-tridecaene, CAS No.: 502-65-8 (Figure 4). 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 standard deviation or relative standard deviation calculated from among-laboratory data. Expressed as the reproducibility standard deviation (SD R ); or % reproducibility relative standard deviation (%RSD R ). 4 Method Performance Requirements See Table 1.

Figure 1. Molecular structure of all- trans α-carotene.

Figure 2. Molecular structure of all- trans β-carotene.

© 2014 AOAC INTERNATIONAL

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. Methods must be capable of resolving lutein from zeaxanthin. 6 Reference Material(s) Neither NIST nor IRMM produce a certified reference material for carotenoids in infant formula. The carotenoid content of SRM 1849a has not been determined (as of November 2014). 7 Validation Guidance Recommended level of validation: Official Methods of Analysis SM . 8 Maximum Time-to-Result No maximum time. Approved by Stakeholder Panel on Infant Formula and Adult Nutritionals (SPIFAN). Final Version Date: September 6, 2014. Effective Date: November 13, 2014.

Table 1. Method performance requirements a Parameter

Minimum acceptable criteria

Analytical range

1–1300 b

Limit of quantitation (LOQ)

≤1 b

Recovery

90–110%

Repeatability (RSD r )

1–100

8% 5%

>100–1300

Reproducibility (RSD R )

1–100

15%

>100–1300 10% a Concentrations apply to: ( a ) ‘ready-to-feed” liquids “as is”; ( b ) reconstituted powders (25 g into 200 g water); and ( c ) liquid concentrates diluted 1:1 by weight. b μg/100 g reconstituted final product; range and LOQ are based on total of cis + trans isomers.

© 2014 AOAC INTERNATIONAL

CAROT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Determination of Carotenoids in Infant Formula and Adult/Pediatric Nutritional Formula using High-Performance Liquid Chromatography with Photo Diode Array Detection.

Martijn Vermeulen, Arjan de Vries, and Fariha Bouraada. TNO Triskelion, Department of Analytical Chemistry, Utrechtseweg 48, Zeist, The Netherlands.

A. Principle Carotenoid esters are saponified using potassium hydroxide in ethanol/water in the presence of sodium ascorbate, sodium sulfide and glycerol to prevent oxidation. Dimethyltocol (DMT) is added as an internal standard and carotenoids are extracted using diisopropyl ether. The extract is subsequently washed and concentrated. Carotenoids are determined by HPLC-UV at 450 nm and DMT is measured at 292 nm. Separation is performed using 2 suplex pkb-100 columns of 25 cm and a gradient of methanol (with ammonium acetate), acetonitrile, isopropyl alcohol and water. B. Apparatus Balances. With readability of 0.1 mg and 0.01 g. Centrifuge. Laboratory centrifuge able to centrifuge 50 mL screw cap (Greiner) tubes. Evaporation device (nitrogen). Zymark TurboVap LV or equivalent. HPLC system. Waters Alliance 2695 HPLC system equipped with a Waters 996 PDA detector (292 nm and 450 nm) and a column oven (20⁰C) or equivalent. LC columns. Supelcosil Suplex pkb-100, pre-column: 4.0x20 mm, 5 µm and two analytical columns in series: 4.6x250 mm, 5 µm or equivalent. Shaker. Laboratory shaker able to shake 250 mL bottles. Spectrofotometer. Spectrofotometer able to measure from 445 – 472 nm using a 1 cm quartz cuvette. C. Reagents and standards Acetonitrile (ACN). HPLC grade, Biosolve 01200701 or equivalent. Ammonium acetate. Merck, art.nr. 1116 or equivalent Butylated Hydroxy Toluene (BHT). Sigma art.nr. B-1378 or equivalent. Carborundum (SiC). Silicon carbide, nr 12, circa 1.44 mm, Boom art.nr. 76054102 or equivalent. beta-Carotene. All-trans, Fluka art.nr.22040 or equivalent. Dichloro methane (DCM). AR, Biosolve art.nr. 13790501 or equivalent. Diisopropyl ether (DIPE). Reinst, stabilized with BHT, Merck art.nr. 800866 or equivalent. Glycerol. Circa 86.5 %, Boom art.nr. 50771 or equivalent. n-Hexane. AR, Biosolve art.nr. 08290501 or equivalent. Isopropyl alcohol (IPA). Baker art.nr. 8068 or equivalent. Lutein. All-trans, Sigma art.nr. X-6250 or equivalent. Lycopene. All-trans, Sigma art.nr. L-9879 or equivalent. Methanol (MeOH). Absolute LC-MS grade, Biosolve art.nr. 13687801 or equivalent. Potassium hydroxide (KOH). P.a., Merck art.nr. 5033 or equivalent. Purified water. Milli-Q or equivalent. Rac-5,7-dimethyltocol (DMT). Matreya art.nr. 1074 or equivalent. Ethanol (EtOH). Absolute, Nedalco 100% technical grade or equivalent. Ultrasonic bath. Ultrasonic bath able to sonicate (glass) tubes. Suction device. Vacuum pump with flask to remove solvent. Water bath. Shaking water bath (85⁰C).

Sodium ascorbate (NaAsc). Merck art.nr. 50076 or equivalent. Sodium chloride (NaCl). P.a., Merck art.nr. 106404 or equivalent. Sodium sulfide (Na 2 Tetrahydrofuran (THF). HPLC grade, Biosolve art.nr. 20220601 or equivalent. Zeaxanthine. All-trans, Fluka art.nr. 14681 or equivalent.

S). Analar Normapur, 58-64 %, VWR art.nr. 83756.290 or equivalent.

CAROT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

D. Preparation of Solutions Ascorbate-sulfide solution. Weigh 16 g of Na 2

S and 40 g of NaAsc into a flask and dissolve in 400 mL water. Add

150 mL of glycerol and mix. Prepare fresh daily. Dissolving solution (ACN/MeOH/DCM, 70/15/20, V/V/V + 0.01 % BHT, m/V). Mix 700 mL of ACN, 150 mL of MeOH and 200 mL of DCM. Add 0.1 g of BHT and dissolve. This solution can be stored at room temperature in a closed container for 1 month. Ethanolic hydroxide 2M. Dissolve 112 g of KOH in 100 ml water and fill to 1 L with EtOH. Prepare fresh daily. Washing solution 5 % KOH. Dissolve 50 g KOH in 1 L water. Washing solution phosphate buffer. Dissolve NaH 2 PO 4 ·H 2 O and Na 2 HPO 4 ·2H 2 O in water (0.2 M, pH 6.7). E. Preparation of Standards Carotenoids are sensitive to UV light and oxygen; conduct all operations using subdued yellow light (Osram HE 28W/62 TL, Philips TLD 36W/16 TL or equivalent) or use amber glassware. Keep all solutions away from direct light. Beta-Carotene stock solution. Into a 100 mL volumetric flask, weigh 10 mg beta-carotene and dissolve in 15 mL DCM. Make up to volume with n-hexane containing 0.1 % (m/V) BHT. Distribute into glass tubes and store in the freezer. To determine the concentration, dilute 500 µL to 20 mL with n-hexane in a volumetric flask and measure the extinction at 453 nm in triplicate in a 1 cm quarts cuvette. Use n-hexane as blank. Calculate the beta-carotene concentration of the stock solution in mg/L as follows: (E x 20 x 10 x 1000) / (E1%453nm x 0.5) where E = the average measured extinction and E1%453nm = 2592. Dimethyltocol (DMT) solution 1 g/L. Dissolve circa 50 mg of DMT in 50 mL of EtOH. Store in the freezer. HPLC standard. Into a glass tube, pipette 100 µL DMT solution, evaporate to dryness and re-dissolve in 2000 µL beta-carotene stock solution. This results in a 2.5 mg/L beta-carotene standard solution. F. Preparation of Samples (1) Weigh sample in duplicate into a 250 mL (Duran) glass bottle; for powder samples 6 and 10 g and for liquid samples 16 and 20 ml. (2) Add 15 mL of the ascorbate-sulfide solution and swirl to dissolve the sample. (3) Add 50 mL of the ethanolic hydroxide solution, 500 µL of DMT solution and some carborundum. (4) Place in a shaking water bath set at 85⁰C for 30 minutes. (5) Cool to room temperature, add 100 ml of DIPE and extract the carotenoids by shaking for 5 minutes using a shaker. (6) Poor 30 mL of the DIPE extract into a 50 mL screw-cap (Greiner) tube, add 20 mL of 5% (m/V) KOH in water and shake for 5 minutes using a shaker. (7) Centrifuge at 1500 g for 5 minutes and remove the water phase (bottom layer) using the suction device. (8) Add 20 mL of phosphate buffer and shake for 5 minutes using a shaker. (9) Centrifuge at 1500 g for 5 minutes, pipette 5000 µL of the extract (top layer) in a glass tube, evaporate to dryness and re-dissolve in 500 µL of ACN/MeOH/DCM (70/15/20 + 0.1 % BHT). Re-dissolve by vortex mixing for 1 minute followed by ultrasonic vibration for 2 minutes and subsequently vortex mixing for 1 minute. Transfer the extract to an HPLC vial. [This extract can also be used for the determination of vitamin A and E and (when vitamin D2 is added) also for the determination of vitamin D3]. G. Analysis (a) Chromatographic conditions. (1) Injection volume. 50 µL. (2) Autosampler temperature. 10⁰C. (3) Column temperature. 20⁰C. (4) Flow rate. 1.5 mL/min. (5) Run time. 41 min. (6) Mobile phases and gradient. A. 7.5 g/L ammonium acetate in MeOH. B. ACN. C. IPA. D. ACN/water (1/1, V/V).

CAROT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Gradient: time (min)

% A 51.1 51.1

% B 36.2 36.2

% C 1.9 1.9 2.0 2.0 1.9 1.9

% D 10.8 10.8

0

10 20 36

54 54

44 44

0 0

36.1

51.1 51.1

36.2 36.2

10.8

41 10.8 (b) System suitability test. Equilibrate the chromatographic system for ≥0.5 h. Inject a working standard solution at least three times and check peak retention times and responses (peak area). Inject working standard solutions on a regular basis within a series of analyses. (c) Calibration. Make single injections of HPLC standard solution at least at the beginning and the end of each analytical series. Establish the calibration curve (one point through zero) by plotting peak response (area) ratios of beta-carotene/DMT versus beta-carotene concentration. Perform linear regression. Calculate the slope (S) of the calibration curve. Other carotenoids are determined using relative response factors. (d) Analysis. Make single injections of sample solutions. (e) Identification. Identify the carotenoid peaks in the chromatograms of the sample solutions by comparison with the retention times and UV spectra of the corresponding peaks in the standard solution. H. Calculations. Summarize peak areas of trans and cis isomers and calculate the content of carotenoids in µg/ 100 g product “as is”, as follows: C = ((Ap / AISp) / (As / AISs)) x (Cs / CIS) x (aIS / w) x RRF x 1000 Where Ap = peak area of the carotenoid in the sample, AISp = peak area of DMT in the sample, As = peak area of the carotenoid in the standard , AISs = peak area of DMT in the standard, Cs = concentration of the carotenoid in the HPLC standard in mg/L, CIS = concentration of DMT in the HPLC standard in mg/L, aIS = amount of DMT added to the sample in µg, w = weight of the sample in g, RRF = relative response factor (see below), and 100 is a conversion factor. Relative response factors of carotenoids relative to beta-carotene are: Lutein = 1.06, zeaxanthine = 0.98, lycopene = 1.21 and alpha-carotene = 0.90. Results A summary of (preliminary) validation results is shown in tables 1 and 2. Calibration was performed with DMT at 50 mg/L and beta-carotene at 0.01 – 10 mg/L. The area-ratio is linear through the intercept with R 2 = 0.9993. Validation results for lutein show that the area-ratio is linear through the intercept from 0.02 – 3.3 mg/L with R 2 = 0.9999. Accuracy of beta-carotene and lutein (total of trans and cis isomers) is between 97 and 109%. Repeatability ranged from 0.9 to 4.5% (RSD r ), and intermediate reproducibility results will be available shortly (RSD R ). Since SRM 1849A does not contain beta-carotene and lutein, the Global control sample from Wyeth was analyzed. Results are: total beta-carotene = 1230 mcg/kg (DV = 3.3%) and total lutein = 1180 mcg/kg (DV = 5.3%). Figure 1 shows an example chromatogram.

CAROT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Table 1. Precision and Spike recovery results for beta-carotene Beta-carotene Performance Analytical range 10 µg/kg – 2.8 mg/kg Limit of quantitation (LOQ) ≤ 10 µg/kg Linearity (10 points) 0.01 – 10 mg/L: r 2 = 0.9993 Repeatability (n = 6-fold, RSD r ) 9.2 µg/kg (SM) 4.5 % 110 µg/kg (SM + spike) 0.9 % 520 µg/kg (IF) 4.2 % 2820 µg/kg (IF + spike) 2.9 % Reproducibility (n = 6 duplicates, RSD R ) 9.2 µg/kg (SM) 110 µg/kg (SM + spike) 520 µg/kg (IF) 2820 µg/kg (IF + spike) Recovery (n = 6) 110 µg/kg (SM + spike) 97 % 2820 µg/kg (IF + spike) 109 %

SM = skimmed milk (liquid). IF = infant formula (powder). Results are based on the total of cis and trans isomers.

Table 2. Precision and Spike recovery results for lutein Lutein

Performance

Analytical range

71 µg/kg – 4.6 mg/kg

Limit of quantitation (LOQ)

≤ 71 µg/kg

0.02 – 3.3 mg/L: r 2 = 0.9999

Linearity (10 points) Repeatability (n = 6-fold, RSD r )

not detectable (SM) 65 µg/kg (SM + spike)

-

2.4 % 2.1 % 2.4 %

1210 µg/kg (IF)

5020 µg/kg (IF + spike) not detectable (SM) 65 µg/kg (SM + spike) 1210 µg/kg (IF) 5020 µg/kg (IF + spike) 65 µg/kg (SM + spike) 5020 µg/kg (IF + spike)

Reproducibility (n = 6 duplicates, RSD R )

Recovery (n = 6)

98 %

101 %

SM = skimmed milk (liquid). IF = infant formula (powder). Results are based on the total of cis and trans isomers.

CAROT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Figure 1a. Example chromatogram of infant formula powder; carotenoids at 450 nm. Trans lutein (Lut) and trans beta-carotene (bCar) are followed by their cis-isomers. Two cis-isomers of lutein co- elute at 16.79 min. DMT elutes at 19.5 min.

Figure 1b. Example chromatogram of a vegetable mixture; carotenoids at 450 nm. DMT elutes at 19.5 min.

AOAC SMPR 2015.002

Table 1. Method performance requirements a Analytical range

20–2000 b

Limit of quantitation (LOQ)

≤20 b

Standard Method Performance Requirements SM (SMPRs) for Total Vitamin B 1 (Thiamin) in Infant and Adult/ Pediatric Nutritional Formula

Recovery

90–110%

Repeatability (RSD r ) Reproducibility (RSD R )

≤5%

≤10% a  Concentrations apply to: (a) “ready-to-feed” liquids “as is”; (b) reconstituted powders (25 g into 200 g water); and (c) liquid concentrates diluted 1:1 by weight. b  μg /100 g reconstituted final product.

Intended Use: Reference Method for Dispute Resolution 1 Applicability Determinations of total vitamin B 1 (thiamin) in all forms of infant, and adult, and/or pediatric formulas (powders, ready-to-feed liquids, and liquid concentrates). Total B 1 defined as the sum of thiamin (CAS No. 70-16-6), thiamin phosphate (CAS No. 532-40-1), thiamin diphosphate (CAS No. 154-87-0), and thiamin triphosphate (CAS No. 3475-65-8). All data should be mass corrected and expressed as thiamin ion. 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Accuracy (corresponds to the VIM definition for “trueness”) .— The closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, 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 standard deviation (%RSD r ).

); or % repeatability relative

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 ). Vitamin B 1 (thiamin) .—2-[3-[(4-Amino-2-methyl-pyrimidin- 5-yl)methyl]-4-methyl-thiazol-5-yl] ethanol. CAS No. 70-16-6 (including all forms listed in applicability statement above). 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) 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 1849 consists of 10 packets, each containing approximately 10 g of material. Certified value of NIST 1849a is 12.57 ± 0.98 mg/kg vitamin B 1 (thiamin). 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: April 17, 2015. Effective Date: April 17, 2015.

© 2015 AOAC INTERNATIONAL

AOAC SMPR 2015.003

Table 1. Method performance requirements a Analytical range

30–4000 b

Limit of quantitation (LOQ)

≤30 b

Standard Method Performance Requirements SM (SMPRs) for Total Vitamin B 2 (Riboflavin) in Infant and Adult/ Pediatric Nutritional Formula

Recovery

90–110%

Repeatability (RSD r ) Reproducibility (RSD R )

≤5%

≤10% a  Concentrations apply to: (a) “ready-to-feed” liquids “as is”; (b) reconstituted powders (25 g into 200 g water); and (c) liquid concentrates diluted 1:1 by weight. b μg/100 g reconstituted final product.

Intended Use: Reference Method for Dispute Resolution

1 Applicability Determinations of total vitamin B 2 (riboflavin) in all forms of infant, adult, and/or pediatric formulas (powders, ready-to-feed liquids, and liquid concentrates). Total B 2 defined as the sum of riboflavin (CAS 83-88-5), riboflavin 5ʹ-phosphate (CAS 146-17-8), and flavin adenine dinucleotide (CAS 146-14-5). All data should be mass corrected and expressed as riboflavin. 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Accuracy (corresponds to the VIM definition for “trueness”). — The closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. Adult/pediatric formula.— Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, 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 standard deviation (%RSD r ).

); or % repeatability relative

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 ). Vitamin B 2 (riboflavin).— An isoalloxazine ring ( flavin ring) substituted at the 10 position with a D-ribityl side chain, and all

forms listed in applicability note above. 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) 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 1849 consists of 10 packets, each containing approximately 10 g of material. Certified value of NIST 1849a is 20.37 ± 0.52 mg/kg riboflavin. 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: April 17, 2015. Effective Date: April 17, 2015.

© 2015 AOAC INTERNATIONAL

AOAC SMPR 2015.004

Table 1. Method performance requirements a Analytical range

200–10000 b

Limit of quantitation (LOQ)

≤200 b

Standard Method Performance Requirements SM (SMPRs) for Total Vitamin B 3 (Niacin) in Infant and Adult/ Pediatric Nutritional Formula

Recovery

90–110%

Repeatability (RSD r ) Reproducibility (RSD R )

≤5%

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 ). Vitamin B 3 (niacin) .—Refers to two compounds: nicotinic acid and nicotinamide. Both forms of the vitamin are equally biological active. Nicotinic acid is chemically pyridine 3-carboxylic acid, CAS No. 59-67-6. Nicotinamide is pyridine 3-carboxcylic acid amide, CAS No. 99586-65-9. 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) 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 1849 consists of 10 packets, each containing approximately 10 g of material. Certified value of NIST 1849a is 109 ± 10 mg/kg niacin. 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: April 17, 2015. Effective Date: April 17, 2015. ≤10% a Concentrations apply to: (a) “ready-to-feed” liquids “as is”; (b) reconstituted powders (25 g into 200 g water); and (c) liquid concentrates diluted 1:1 by weight. b μg/100 g reconstituted final product.

Intended Use: Reference Method for Dispute Resolution 1 Applicability Determinations of total vitamin B 3 (niacin) in all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). Total B 3 defined as the sum of nicotinic acid (CAS No. 59-67-6) and nicotinamide (CAS No. 99586-65-9), and reported as niacin. 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Accuracy (corresponds to the VIM definition for “trueness”) .— The closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, 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 ).

© 2015 AOAC INTERNATIONAL

AOAC SMPR 2015.005

Table 1. Method performance requirements a Analytical range

10–2000 b

Limit of quantitation (LOQ)

≤10 b

Standard Method Performance Requirements SM (SMPRs) for Total Vitamin B 6 (Pyridoxine) in Infant and Adult/ Pediatric Nutritional Formula

Recovery

90–110%

Repeatability (RSD r ) Reproducibility (RSD R )

≤5%

≤10%

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

Intended Use: Reference Method for Dispute Resolution 1 Applicability Determinations of total vitamin B 6 (pyridoxine) in all forms of infant, adult, and/or pediatric formula (powders, ready-to-feed liquids, and liquid concentrates). Total B 6 defined as the sum of pyridoxine(CAS No. 65-23-6), pyridoxal (CAS No. 66-72-8), pyridoxamine (CAS No. 85-87-0), and the 5' phosphorylated forms, pyridoxal 5' phosphate (CAS No. 54-47-7) and pyridoxamine 5' phosphate (CAS No. 3475-65-8). All data should be mass corrected and expressed as pyridoxine. 2 Analytical Technique Any analytical technique that meets the following method performance requirements is acceptable. 3 Definitions Accuracy (corresponds to the VIM definition for “trueness”). — The closeness of agreement between the average of an infinite number of replicate measured quantity values and a reference quantity value. Adult/pediatric formula .—Nutritionally complete, specially formulated food, consumed in liquid form, which may constitute the sole source of nourishment, 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 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 ). Vitamin B 6 (pyridoxine) .—All 2-methyl-3-hydroxy-5-hydroxy methyl pyridine compounds with equivalent biological activity of pyridoxine (PN) (2-methyl-3-hydroxy-4,5-bis(hydroxymethyl)- pyridine in rats. See applicability statement above (CAS No. for pyridoxine: 65-23-6). 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) 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 1849 consists of 10 packets, each containing approximately 10 g of material. Certified value of NIST 1849a is 13.46 ± 0.93 mg/kg vitamin B 6 (pyridoxine). 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: April 17, 2015. Effective Date: April 17, 2015.

© 2015 AOAC INTERNATIONAL

B-VIT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

I. SUMMARY Water-soluble vitamins in infant formula or adult nutritional samples are extracted with a mixture of mild acid-Methanol. Quantitation of the extracted solution is carried out via LC-MS/MS using ESI in the positive mode. a. Intended use of method: The method is applicable for the concurrent quantitative analysis of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin present in milk- and soy- based infant formula and adult nutritionals and formulas containing hydrolyzed protein. b. Critical components: Correct personal and environmental safety standards shall be used while performing this analytical method. Laboratory personnel handling solvents, acids and reagents should be knowledgeable of their potential hazards. Consult the Material Safty Data Sheets for information on the hazards and take proper precautions.Transfer solvents and acids inside efficient fume hoods and extractors. Ensure all glassware is free from chipping and hairline cracks. II. EQUIPMENT & APPARATUS Equivalents may be used unless otherwise specified.

Equipment LC-MS/MS

Description (Brand, make, model, etc.)

Agilent UHPLC 1290 series- ABSCIEX QTRAP 6500 ESI Positive mode or equivalent.

HPLC Column

Shishedo, CAPCELL PAK C18 column, MGIII-H, 100mm x 2.0mm x 3 μm or equivalent.

Vortex mixer Ultrasonic bath

Labnet VX100 or equivalent. Ultrasonik 208H or equivalent.

High speed centrifuge

Hettich Rotina 420R, with rotor for 2mL centrifuge tubes and capable of 15000 rpm at 4  C or equivalent. Mettler Teledo XP204, capable of weighing to four decimal places or equivalent. Agilent Technologies, amber, 2mL capacity, with crimp caps and PTFE seals. 50 ml PP, Amber Falcon tube with screw cap or equivalent. Memmert or equivalent. EYELA Multishaker MMS or equivalent Eppendorf, 10-100 µl, 100-1000 µl and 1-5 ml or equivalent. Witeg, class A: 2, 3, 4, 5, 10, 15, 25 ml or equivalent. Witeg, class A, amber: 1 (clear), 10, 100, 200, 1000 ml or equivalent. Filtrex, 0.2 µm, 13 mm or equivalent and MSF filter 0.2 µm, 47mm or equivalent. 2 ml PP, Amber, Axygen or equivalent.

Hot-Air oven (105  C) Analytical balance

Mechanical Shaker

HPLC vials

Centrifuge tube

Microcentrifuge tube Auto pipette with tip Volumetric pipet Volumetric flask Syringe Filter Nylon membrane

Page 1

PAGE 1 OF 13

B-VIT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

CHEMICALS & REAGENTS Equivalents may be used unless otherwise specified. Standard and reagent preparations may be scaled up or down as needed. No. Reagent (specify grade, if applicable) Concentration Preparation No. Brand Shelf life Storage 1 Formic acid (CH 2 O 2 ), AR 98% N/A Merck as per COA Room temperature 2 Hydrochloric Acid (HCl), AR 37% N/A Merck as per COA Room temperature 3 Hepafluorobutyric acid (HFBA) 98% N/A Sigma as per COA Room temperature 4 Methanol (MeOH; CH 3 OH), HPLC Grade 100% N/A J.T. Baker as per COA Room temperature 5 Acetronitrile (ACN; CH 3 CN) 100% N/A J.T. Baker as per COA Room temperature 6 Ammonium formate, AR ≥ 99% N/A Fluka as per COA 2-8  C 7 Ammonium hydroxide (NH 4 OH), AR 28-30% N/A Merck as per COA Room temperature 8 Isotope Thiamine chloride (Vitamin B1 HCl) (4,5,4-Methyl-13C3, 99%) (*C3C9H17ClN4OS) ≥ 98% N/A CIL Until exhausted or evidence of contamination -20 to-30  C Protect from light, air and moisture.

9

Isotope Riboflavin (Vitamin B2) (13C4, 99%; 15N2, 98%) (C13*C4H20N2*N2O6) Isotope Nicotinamide (D4, 98%) (C6D4H2N2O)

N/A

CIL

Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of

Room temperature away from

≥ 98%

light and moisture

10

N/A

CIL

Room temperature away from

≥ 98%

light and moisture

11

Isotope Nicotinic acid (D4, 98%) (C6D4HNO2)

N/A

CIL

Room temperature away from

≥ 98%

light and moisture

12

Isotope Pantothenic acid, Calcium salt monohydrate (beta-alanyl-13C3, 99%; 15N, 98%) Isotope Pyridoxine HCl (4,5-BIS(hydroxyl methyl)-13C4, 99%) (*C4C4H11NO3.HCl) Isotope Pyridoxal:HCl (Methyl-D3, 98%) (C8D3H6NO3.3HCl) Isotope Pyridoxamine:2HCl (Vitamin B6) (Methyl-D3,

N/A

CIL

-5 to 5  C and desiccated. Protect from light. -20 to-30  C Protect from light, air and moisture. Room temp away from

≥ 98%

13

N/A

CIL

≥ 98%

14

N/A

CIL

≥ 98%

light and moisture

15

N/A

CIL

-20  C Protect from light.

≥ 98%

Page 2

PAGE 2 OF 13

B-VIT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

98%) (C8D3H9N2O2.2HCl) Isotope Biotin (chemical purity 97%) (6,6-D2, 98%) (C10D2H14N2O3S) Thiamine Hydrochloride (Vitamin B1 ) (C12H17ClN4OS-HCl) Riboflavin (Vitamin B2) (C17H2ON4O6) Calcium pantothenate (C18H32CaN2O10) Pyridoxine hydrochloride (C8H11NO3. HCl) Pyridoxal hydrochloride (C8H9NO3.HCl) Pyridoxamine dihidrochloride (C8H12N2O2. 2HCl) Biotin (C10H16N2O3S) Deionized water (DI) Resistivity ≥ 18.2 M  75 mM Formic acid-20% MeOH mixture solution Nicotinamide (C6H6N2O), USP Nicotinic acid (C6H5NO2) Thiamine Cl (Vitamin B1) stock internal standard solution Riboflavin (Vitamin B2) stock internal standard solution Nicotinamide stock internal standard solution Nicotinic acid stock internal standard solution Pantothenic acid stock internal standard 1 N HCl 50 mM HCl 1 % Formic acid

contamination

16

97%

N/A

CIL

Until exhausted or evidence of contamination See remark below See remark below See remark below See remark below See remark below See remark below as per COA

Room temp away from

light and moisture

17

99.8%

N/A

USP

2-8  C

18

100 %

N/A

USP

2-8  C

19

99.9%

N/A

USP

2-8  C

20

99.8%

N/A

USP

2-8  C

21

99%

N/A

USP

2-8  C

22

99.5%

N/A

USP

2-8  C

23

N/A

Sigma- Aldrich Sigma- Aldrich

-20 to-30  C

≥ 99 %

24

N/A

as per COA

-20 to-30  C

≥ 98 %

25

99.3%

N/A

USP

See remark below

2-8  C

26

Fresh

27

1

1 year

Room temperature Room temperature Room temperature Room temperature

28

2

1 year

29

3

1 year

30

4

1 year

31

20 mcg/ml

5

Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or

2-8  C

32

20 mcg/ml

6

2-8  C

33

270 mcg/ml

7

2-8  C

34

25 mcg/ml

8

2-8  C

35

30 mcg/ml

9

2-8  C

Page 3

PAGE 3 OF 13

B-VIT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

solution

evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination Until exhausted or evidence of contamination

36

Pyridoxine stock internal standard solution

32 mcg/ml

10

2-8  C

37

Pyridoxal hydrochloride stock internal standard solution Pyridoxamine dihydrochloride stock internal standard solution Biotin stock internal standard solution Mixed intermediate internal standard solution I Thiamine HCl stock standard solution Riboflavin stock standard solution Nicotinamide stock standard solution Nicotinic acid stock standard solution Pantothenic acid stock standard solution Pyridoxamine stock standard solution Biotin stock standard solution Mixed water-soluble vitamin intermediate standard solution I Mixed water-soluble vitamin intermediate standard solution II Mixed water-soluble vitamin intermediate standard solution III Mixed water-soluble vitamin working standard solution 1-16 Mobile Phase A: 0.04% HFBA-0.05% Formic Pyridoxine stock standard solution Pyridoxal stock standard solution

25 mcg/ml

11

2-8  C

38

20 mcg/ml

12

2-8  C

39

24 mcg/ml

13

2-8  C

40

14

2-8  C

41

200 mcg/ml

15

3months

2-8  C

42

50 mcg/ml

16

3months

2-8  C

43

1000 mcg/ml

17

3months

2-8  C

44

100 mcg/ml

18

3months

2-8  C

45

500 mcg/ml

19

3months

2-8  C

46

174 mcg/ml

20

3months

2-8  C

47

100 mcg/ml

21

3months

2-8  C

48

73 mcg/ml

22

3months

2-8  C

49

100 mcg/ml

23

3months

2-8  C

50

24

3months

2-8  C

51

25

1month

2-8  C

52

26

1month

2-8  C

53

27

Freshly prepared

54

28

2 weeks

Room temperature

Page 4

PAGE 4 OF 13

B-VIT-01 FOR ERP/WORKING GROUP USE ONLY DO NOT DISTRIBUTE

Simultaneous Determination of Thiamine HCl, Riboflavin, Niacin (Nicotinic Acid and Nicotinamide), Pantothenic acid, Vitamin B6 (Pyridoxine, Pyridoxal and Pyridoxamine) and Biotin in Infant Formula and Adult Nutritionals

CONFIDENTIAL METHOD FOR ERP ASSESSMENT ONLY

acid-5mM Ammonium formate

Remark: USP standard expiry information can be obtained using the following website: http://www.usp.org/reference-standards?gclid=CNaTqtu4wMUCFRcOjgodZXcAog

Preparation No. Procedure 1

75 mM Formic acid-20% MeOH mixture solution : 3.11 mL Formic acid and 200 mL MeOH in a 1000mL volumetric flask. Adjust and make up to volume with water. 1 N HCl : 83.33 mL of 37%HCl diluted to 1000mL with water. 50 mM HCl : 25 mL of 1 N HCl diluted to 500 mL with water. 1 % Formic acid : 5 mL Formic acid diluted to 500 mL with water. Weight 0.001x g IS Thiamin Cl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Riboflavin, dissolved and diluted to 50 mL with water. Weight 0.01xx g IS Nicotinamide, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Nicotinic acid, dissolved and diluted to 50 mL with water. Weight 0.0015 g IS Pantothenic acid, Calcium salt monohydrate, dissolved and diluted to 50 mL with water. Weight 0.0015 g IS Pyridoxine HCl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Pyridoxal HCl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Pyridoxamine.2HCl, dissolved and diluted to 50 mL with water. Weight 0.001x g IS Biotin, dissolved and diluted to 50 mL with water. Pipet each stock internal standard solution into 100 mL volumetric flask as follows: 4 ml stock IS Thiamine HCl 8 ml stock IS Riboflavin DryThiamine HCl at 105 ± 5 ◦ C 2 hours. Weight 0.01xx g Thiamine HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Dry Riboflavin at 105 ± 5 ◦ C 2 hours. Weight 0.005x g Riboflavin, dissolved and diluted with 1 % Formic acid in 100 mL volumetric flask. Dry Nicotinamide in dessicator approx. 4 hours. Weight 0.05xx g Nicotinamide, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Nicotinic acid, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Dry Calcium pantothenate at 105 ± 5 ◦ C 3 hours. Weight 0.025x g Calcium pantothenate, dissolved and diluted with water in 50 mL volumetric flask. Weight 0.01xx g Pyridoxine HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Pyridoxal HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Pyridoxamine.2HCl, dissolved and diluted with 50 mM HCl in 50 mL volumetric flask. Weight 0.005x g Biotin, dissolved with water and 1 ml NH4OH, diluted with water in 50 mL volumetric flask. 2 ml stock IS Nicotinamide 15 ml stock IS Nicotinic acid 10 ml stock IS Pantothenic acid 0.5 ml stock IS Pyridoxine 0.5 ml stock IS Pyridoxal 0.6 ml stock IS Pyridoxamine 0.8 ml stock IS Biotin dissolved and diluted to 100 mL with water.

2 3 4 5 6 7 8 9

10 11 12 13 14

15

16

17

18

19

20

21

22

23

24

Pipet stock standard solution into 50 ml volumetric flask as follows : 2.5 ml 100 mcg/ml Thiamine HCl stock standard solution 15 ml 100 mcg/ml Riboflavin stock standard solution

Page 5

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