Review Team (February 2016)

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

REVIEW TEAM MEETING

February 22, 2016

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

UNITED STATES dboyd@aoac.org 301.924.7077 x126

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Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

Applicable for the determination of amino acids (including Taurine) in all forms of infant, adult, and/or pediatric formula (powders, ready to feed liquids and liquid concentrates). The LOQs for the method ranged from 2.0 to 10000 mg/100g. Caution: Refer to Material Safety Data Sheet (MSDS) of chemicals prior to use, and follow the suggested personal protective equipment. A. Principle: This procedure involves the conversion of proteins present in the foods to amino acids by different hydrolysis methods (see table 1) based on the amino acids chemical nature. The resulting amino acids are derivatized using 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) reagent (except Tryptophan) to form stable urea derivatives of each amino acid. The derivatized forms of amino acids are separated by UHPLC and quantitated by Diode Array Detector (Tryptophan by Fluorescence detector) using Norvaline as internal standard. Table 1: Different Hydrolysis Procedures and amino acids to be determined in each method Procedure Amino acids Acid Hydrolysis Histidine, Taurine, Serine, Arginine, Glycine, Aspartic acid (sum of Aspartic acid and Asparagine), Glutamic acid (sum of Glutamic acid and Glutamine), Threonine, Alanine, Proline, Lysine, Tyrosine, Valine, Isoleucine, Leucine, Phenyl alanine. Oxidative Acid Hydrolysis Cysteine a (sum of cysteine and cystine), Methionine b Alkaline Hydrolysis Tryptophan c a, b Cystine and Methionine are converted into Cysteic acid and Methionine sulfone by performic B. Apparatus: a) Liquid Chromatograph: Agilent UHPLC Infinity series with Binary Solvent Manager capable of 1200 bar pressure. b) Column: AccQ.Tag TM Ultra, C18, 1.7 μm, 2.1 x 100 mm (Waters Part No. 205000343). c) Detector: Agilent Infinity series UV/DAD detector with 1 μL flow cell and Fluorescence detector. d) Injector: Agilent Infinity Series Auto sampler with column oven. e) Data System: Agilent Open lab CDS. f) Centrifuge. g) Hot air oven: Capable of 110 ± 2°C h) Turbo-vap evaporator: Concentration evaporator workstation (Biotage) i) pH meter. j) Digestion Vessels: 20 mL glass vials with relse cap (crimp caps and Teflon lined septas). k) Vortex mixer l) Syringe filters: 0.22 μm, PTFE. m) Total Recovery Vials: Waters (Part No.: 186000384C) C. Reagents: a) AccQ-Tag Eluent A concentrate: Waters, Part No: 186003838 b) AccQ-Tag Ultra Derivatization kit: Waters, Part No: 186003836. This kit contains 5 sets of vials and each set contains 3 vials labeled with 1 (Borate Buffer), 2A (Reagent Powder), 2B (Reagent Diluent). c) De-ionized laboratory water - ≥ 18 Mohm/cm acid oxidation prior to acid hydrolysis. c Determined by Fluorescence detector

d) Acetonitrile – HPLC grade. e) Methanol – HPLC grade.

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Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

f) Hydrochloric acid, 35% - AR grade . g) Ortho-Phosphoric acid, 85%- AR grade. h) Hydrogen peroxide, 30% v/v. i) Formic acid, 99% - AR & HPLC grade. j) Ethanolamine – HPLC grade. k) 1,1,1-trichloro-2-methyl-2-propanol – AR grade. l) Phenol – AR grade. m) Sodium Hydroxide – AR grade. D. Standards:

a) Amino acids standard, Hydrolysate, 2.5 mM concentration of each amino acid except Cystine(1.25mM) : Waters, Part No.: WAT 088122, (contains Aspartic acid, Serine, Glutamic acid, Glycine, Histidine, Arginine, Threonine, Alanine, Proline, Cystine, Tyrosine, Lysine, Valine,

Methionine, Isoleucine, Leucine and Phenyl Alanine). b) Tryptophan : Sigma, product No.: T0254 or equivalent). c) Taurine : Sigma, product No.: T0625 or equivalent). d) Cysteic acid: Sigma, product No.: 30170 or equivalent). e) Methionone Sulfone : Sigma, product No.: M0876 or equivalent).

f) Norvaline: Sigma, product No.: N7627 or equivalent). g) Standard Reference Material – NIST SRM 1849a. E. Solutions Preparation: a) Hydrochloric acid, 6M : Add 53 mL of Hydrochloric acid to 47 mL of water in a glass beaker. Cover the beaker with a watch glass and boil for 2 minutes in a fume hood. b) Hydrochloric acid, 20 mM: Add 1.7 ml of 6M Hydrochloric acid to 500 mL laboratory water. c) Mobile Phase A1: Quantitatively transfer 25 ml of Eluent A concentrate into a 500 mL volumetric flask, add approximately 200 mL laboratory water to dissolve and fill to volume with laboratory water. d) Mobile Phase B1 : Add 6.5(±0.1) mL Formic acid (HPLC grade) to 500 mL Acetonitrile and dissolve. e) Mobile Phase A2: Dissolve 2.85 mL acetic acid in 900 ml laboratory water and add 50 mL methanol containing 0.5g of 1,1,1-trichloro-2-methyl-2-propanol. Adjust to pH 5.0 with Ethanolamine and makeup to 1000 ml. f) Performic acid solution: Dissolve 0.24g of phenol in 45 mL of formic acid. Add 5 mL of 30% v/v Hydrogen peroxide and mix gently. Incubate the solution between 20 - 30 ºC for 1 hour to form performic acid, then cool in an ice bath for 15 minutes before adding to the samples. g) Reconstitution of Derivatizing agent from Derivatization kit: Transfer 1 mL of diluent from vial 2B to vial 2A. Cap the vial tightly and vortex for 2 minutes. Heat on a preheated water bath at 55°C for 5 minutes. h) Sodium Hydroxide solution, 4M: Dissolve 16g sodium hydroxide pellets in 80 mL laboratory water. Cool and dilute to 100 mL with laboratory water. i) Orthophosphoric acid, 0.5M: Add 3.4 mL of orthophosphoric acid to 80 mL laboratory water and make upto 100 mL. j) Hydrochloric acid, 1.0M: Add 16.4 mL of hydrochloric acid (6M) to laboratory water and make upto 100 mL with water. k) Standards Preparation: 1) Preparation of Stock Standards: i) Amino acids standard, Hydrolysate, 2.5mM: Transfer the content from the ampoule into a new HPLC vial, close tightly and store at -20°C±2°C for 3 months. ii) Taurine Stock standard preparation, 2.5 mM: Accurately weigh 15.64 mg of Taurine into a 50 mL standard volumetric flask. Dissolve and makeup to 50 mL with laboratory water. Store at -20°C±2°C for 2 months.

2

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography iii) L-Cysteic acid, 2.5mM: Accurately weigh 23.40 mg of L-Cysteic acid monohydrate into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at -20°C±2°C for 2 months. iv) L-Methionine Sulfone, 2.5 mM: Accurately weigh 22.65 mg of L-Methionine Sulfone into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at -20°C±2°C for 2 months. v) Tryptophan stock standard, 500 mg/L: Accurately weigh 25.0 mg of tryptophan standard into a 50 ml standard volumetric flask and dissolve in 0.1 M HCl and makeup to the mark. vi) Norvaline, stock Internal Standard Preparation, 2.5 mM: Accurately weigh 14.64 mg of Norvaline into a 50 ml standard volumetric flask. Dissolve and makeup to 50 ml with water. Store at -20 °C±2°C for 2 months. (Note: Apply appropriate Purity, Salt and Moisture corrections to the stock standards concentrations) 2) Preparation of Intermediate, working standards and derivatization procedure for Acid

Hydrolysis and Oxidative Acid Hydrolysis methods: i) Table 2: Intermediate standards (IS) preparation:

Stock std. Conc. (mM)

Volume of Stock std. (ml)

Volume of diluent c (ml)

Intemediate std. Conc. (mM)

Intermediate std code

Stock Std. Name

Amino acids, Hydrolysate

2.5

0.40

0.20

1.0

IS Mix - 1

Taurine

2.5

0.40

L-Cysteic acid L-Methionine Sulfone

2.5

0.40

1.0

IS Mix - 2

0.20

2.5

0.40

Norvaline

2.5

0.10

0.90

0.25

--

Norvaline

0.25

0.20

0.80

0.05

--

c Diluent – Laboratory water Storage and Validity: - 20 ± 2˚C for 2 m onths ii) Table 3: Working standards preparation:

Vol. of working Std. (mL)

Final Volume (mL)

Working std. Conc. (mM)

IS Mix std. d Conc. (mM)

Vol. of 2.5 mM Norvaline (mL)

Vol. of Diluent* (mL)

1.00 1.00 1.00 0.50 0.10

0.50 0.25 0.10 0.10 0.10

0.10 0.10 0.10 0.10 0.10

0.40 0.65 0.80 0.80 0.80

1.00 1.00 1.00 1.00 1.00

0.50 0.25 0.10 0.05 0.01

d Choose appropriate IS Mix standard based on the hydrolysis procedure. iii) Preparation of Calibration standards by derivatization of working standards: Quantitatively transfer 10 μL of each working standard into total recovery vials. Add 70 μL of borate buffer to each vial and vortex for 30 seconds. Add 20 μL of reconstituted AccQ.Tag Ultra reagent powder and vortex for 30 seconds. Let it stand for 1 minute at

3

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography room temperature and heat in a preheated water bath at 55 ˚C for 5 minutes. Final concentrations of calibration standards are 1.0, 5.0, 10.0, 25.0 and 50.0 μM respectively. 3) Preparation of calibration standards for Tryptophan (alkaline hydrolysis method): Table 4: Stock std. Conc. (mg/L) Vol. of stock Std. (mL) Makeup Volume (mL) Calibration std. Conc.(mg/L) 500.0 1.00 10.0 50.0 500.0 0.50 10.0 25.0 500.0 0.20 10.0 10.0 50.0 1.00 10.0 5.00 50.0 0.20 10.0 1.00 1.0 1.00 10.0 0.10 1.0 0.20 10.0 0.02

4

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

F. Procedure

a) Acid Hydrolysis procedure: 1) Accurately weigh 0.5 g of finely ground sample into a digestion vessel. 2) Add a speck of phenol (Approx. 10 mg) and 5mL of 6M HCl. 3) Purge slowly with nitrogen on top of the vial solution to remove all the atmospheric oxygen. 4) Immediately seal with relse cap using crimper and ensure the sealing is tight. 5) Keep the sealed vials in a hot air oven, initially at room temperature. 6) Raise the hot air oven temperature to 110°C±5°C and hydrolyze the samples for 20 hours. 7) Cool the vials to room temperature and quantitatively transfer the contents into a 50 ml standard volumetric flask. Makeup the flask with laboratory water and mix contents. 8) Filter a portion of the sample solution through 0.22 μm PTFE filters into a 15 ml disposable centrifuge tube. 9) Dilute the filtered aliquots further, depending on the protein content present in the samples (See Table 5) 10) Take 100 μL of the final diluted sample into the bottom of a micro centrifuge tube and add 50 μL of 0.05mM Norvaline internal standard.

11) Dry the tubes under gentle stream of nitrogen at 40°C. 12) Proceed for derivatization with the dried sample tubes. Table 5: Protein % Sample weight (g) Makeup Vol. (ml)

Volume to be taken for drying (μL)

Further dilutions

< 5.0

0.50 0.50 0.50 0.50

50.0 50.0 50.0 50.0

1.0 ml to 10 ml 0.5 ml to 10 ml 0.75 ml to 25 ml 1.0 ml to 50 ml

100.0 100.0 100.0 100.0

5.0 to 20.0 20.0 to 40.0 40.0 to 60.0

b) Oxidative acid hydrolysis procedure: 1) Accurately weigh 0.5 g of finely ground sample into a digestion vessel. 2) Add 3 to 4 mL of cold performic acid solution and ensure the sample is completely dispersed. 3) Keep in a refrigerator at 4 ˚C for 16 hours. 4) Evaporate residual performic acid solution under nitrogen at 50˚C 5) Add 5 mL of 6M Hydrochloric acid and proceed as per the acid hydrolysis procedure (from step 3 to 12). c) Derivatization Procedure: 1) Add 20 μL of 20 mM Hydrochloric acid solution to the bottom of the dried sample tube and vortex for 2 minutes. 2) Add 60 μL of Borate buffer (Vial 1 from derivatization kit) and vortex for 30 seconds. 3) Add 20 μL of derivatizing agent and vortex for 30 seconds. 4) Let it stand for 1 minute at room temperature and heat at 55°C in a preheated water bath for 5 minutes. 5) Vortex the contents in the whole tube and centrifuge the tube at 2000 rpm for 2 minutes to bring down all the liquid to the bottom of the vial. 6) Transfer the derivatized sample solution into total recovery vials and inject 1μL into UHPLC- DAD. d) Alkaline hydrolysis procedure: 1) Accurately Weigh 0.5 g of finely ground sample into a digestion vessel. 2) Add 5 mL of 4N Sodium Hydroxide solution

3) Seal the vial with relse cap using crimper and ensure the sealing is tight. 4) Keep the sealed vials in a hot air oven, initially at room temperature.

5

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

5) Raise the hot air oven temperature to 110°C±5°C and hydrolyze the samples for 20 hours. 6) Cool the vials to room temperature and quantitatively transfer the contents into a 50 ml polypropylene centrifuge tubes. 7) Add 5 mL of 0.5M Orthophosphoric acid and adjust the pH of the solution to 3.0 with 1M Hydrochloric acid 8) Quantitatively transfer the contents to 50 mL standard volumetric flask. 9) Add 10 mL of methanol to the flask and makeup to the mark with laboratory water. 10) Mix well and let it stand for 10 minutes at room temperature. 11) Take 1 mL of the supernatant into a 10 mL standard volumetric flask and makeup to the mark with water. 12) Filter through 0.22 μm membrane filter and inject 2μL into UHPLC-FLD. e) Chromatographic Conditions: 1) Chromatographic conditions for Acid Hydrolysis method: Table 6: Column AccQ.Tag Ultra C18, 1.7 μM, 2.1 x 100 mm Mobile phase A Mobile Phase A1 Mobile phase B Mobile Phase B1 Flow rate 0.7 mL/minute Injection Volume 1.0 μL Column Temperature 55°C Detector Wavelength 260 nm Total Run Time 12.5 minutes Gradient Method Time (minutes) A % B% 0.00 99.90 0.10 0.54 99.90 0.10 2.50 98.70 1.30 3.00 98.00 2.00 4.03 96.00 4.00 4.83 93.90 6.10 5.13 93.10 6.90 5.48 92.00 8.00 5.74 90.90 9.10 8.74 78.80 21.20 9.04 40.40 59.60 9.05 10.00 90.00 9.64 10.00 90.00 9.73 99.90 0.10 12.5 99.90 0.10

2) Chromatographic conditions for Oxidative Acid Hydrolysis method: Table 7:

6

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

Column

ACCQ-TAG ULTRA C18 1.7μM, 2.1 x 100mm

Mobile phase A Mobile phase B

Mobile Phase A1 Mobile Phase B1 0.6 mL/minute

Flow rate

Injection Volume

1.0 μL 50 °C 260 nm

Column Temperature Detector Wavelength

Run Time

14 minutes Gradient Method

Time (minutes)

A %

B%

0.00 0.63 2.33 2.92 3.50 4.12 4.70 5.64 5.99 6.16 6.39 6.70 9.03

99.90 99.90 99.50 98.70 98.00 97.00 96.00 93.90 93.10 92.60 92.00 90.90 78.80 40.40 10.00 10.00 99.90 99.90

0.10 0.10 0.50 1.30 2.00 3.00 4.00 6.10 6.90 7.40 8.00 9.10

21.20 59.60 90.00 90.00

10.00 10.01 11.01 11.02 14.00

0.10

0.10 3) Chromatographic conditions for Alkaline Hydrolysis method: Table 8: Column

Agilent Zorbax RRHD, 1.8μm, 2.1 X 50 mm

Mobile phase

Mobile Phase A2 0.2 mL/minute

Flow rate

Injection Volume

2.0 μL

Column Temperature Detector Wavelength

25°C

Ex. 280nm, Em. 356nm

Run Time

10minutes

Pump Mode

Isocratic

7

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Amino Acids in Infant Formula and Adult/Pediatric Formulas Ultra High Performance Liquid Chromatography

f) Calculations:

Area of std. * Conc. of ISTD in μM

Relative Response factor (RRF) =

ISTD Area * Conc. of std. in μM

- A b

) * W AA

* C ISTD

* V s

* DF S

(A s

Amino acid (g/100g) =

* 1000 * W s

* 10000

RRF * A ISTD

= Sample area = Blank area

A s A b

= Molecular weight of corresponding amino acid.

W AA A ISTD C ISTD

= ISTD area of sample. = Conc. of ISTD in μM = Sample Makeup Volume = Sample weight in grams

V s W s

= Sample dilution factor (Other than V s and W s )

DF S

1000 = Conversion factor for μM to mM 10000 =Conversion factor for mg/kg to g/100g

G. References: a) UPLC Amino acid analysis solution system guide. Waters

b) ISO 13904:2005(E) : Animal Feeding stuffs – Determination of Tryptophan content. c) ISO 13903:2005- : Animal Feeding Stuffs – Determination of Amino acids Content. d) Sulfur amino acids in food, feed ingredients and processed foods – AOAC 985.28 e) Amino acids in Feeds – AOAC 994.12

8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Table: Method Performance requirements for Amino acids as per SMPR 2014.013

Parameter

Minimum acceptable criteria

Analytical Range

0.4 to 2500 mg/100g

3.6 - 45.0 45.0 - 450 ± 8 % 450 - 22500 ± 5 % 3.6 - 45.0 ≤ 4 % 45.0 - 450 ≤ 3 % 450 - 22500 ≤ 2 % 3.6 - 45.0 ≤ 6 % 45.0 - 450 ≤ 4 % 450 - 22500 ≤ 3 % 3.6 mg/100g on as is basis ± 10 % 3.6 to 22500 mg/100g

≤ 0.4 mg/100g on reconstituted powder basis

Limit of quantification (LOQ) on reconstituted product

5.0 -50 0.4 - 5.0 50 - 2500

± 10 %

Recovery

± 8 % ± 5 % ≤ 4 % ≤ 3 % ≤ 2 % ≤ 6 % ≤ 4 % ≤ 3 %

0.4 -50 5.0 - 50

Repeatability (RSDr)

50 - 2500 0.4 - 5.0

Reproducibility (RSD R )

5.0 - 50

50 - 2500

AMINO ACIDS METHOD VERIFICATION AGAINST AOAC SMPR 2014.013

NIST SRM 1849a Results

Repeatability

Reproducibility

SMPR Recovery limits b , %

SMPR Recovery limits b , %

NIST SRM 1849a Reference values(mg/100g)

SMPR RSD R limits b , %

SMPR RSDr limits b , %

Result a (mg/100g)

Recovery %

Amino acid

Result (mg/100g) Recovery % RSD R %

RSDr %

Method of Analysis

L-Threonine L-Glutamic acid L-Aspartic acid L-Glycine L-Argenine L-Serine L-Taurine L-Histidine

319.7

92 - 108 90 - 110 95 - 105 92 - 108 92 - 108 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 NA

≤ 3 ≤ 4 ≤ 2 ≤ 3 ≤ 3 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2 ≤ 2

316.1

100.3 102.9

2.4 4.9 2.2 3.7 2.9 3.3 2.5 1.7 2.7 1.8 4.0 3.8 1.9 2.0 1.4 2.1 3.5

92 - 108 90 - 110 95 - 105 92 - 108 92 - 108 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 95 - 105 NA

≤ 4 ≤ 6 ≤ 3 ≤ 4 ≤ 4 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3 ≤ 3

315

101.5

2.3 2.4 1.3 1.6 3.2 2.2 2.0 0.9 1.5 0.7 2.1 2.6 1.6 1.1 1.2 0.6 2.3

34.5

37.7

37

94.4 94.9 92.4 95.5

683.4 369.4 230.1

706.3 378.9 234.0

98.1 94.7 97.1

720 400 241

1099.5 2761.3

1082.4 2725.6

101.2 105.2

1070 2590

102.8 106.6

619.1 445.5

613.5 442.0

95.9 97.1 96.7

640 455

96.7 97.9 98.2

Acid Hydrolysis followed by UHPLC-DAD determination

L-Lysine L-Proline L-Tyrosine L-Alanine

1173.8 1068.7

1155.2 1064.6

1195 1010

105.4

105.8

476.6 440.3 781.1 694.2

471.3 429.3 772.0 694.6

92.4

510

93.4

L-Phenyl Alanine L-Leucine L-Isoleucine L-Valine L-Methionine

NA

NA

NA

101.6 105.2

760 660

102.8 105.2

1243.6

1219.8

96.7 95.2

1261

98.6 96.8

561.3

552.2

580

Alkaline hydrolysis followed by UHPLC-FLD determination Performic acid oxidation followed by acid hydrolysis and UHPLC-DAD determination

Cysteine + Cystine

138.4

107.6

1.6

92 - 108

≤ 3

131.8

102.5

4.2

92 - 108

≤ 4

128.6

L-Methionine

564.3

NA

≤ 2

--

--

--

--

--

NA

NA

1.7

L-Tryptophan

184

173.3

--

92 - 108

≤ 3

--

--

--

92 - 108

≤ 4

96.8

a Results reported are as is sample basis b SMPR limits are on 'reconstituted sample basis' and converted into 'as is sample basis' by multiplying with a factor 9

Prepared By: BKVR Prasad

Reviewed By: Meena Mariappan

Asst. Manager

General Manager - Chemical Testing

Product Service-COE TUV-SUD South Asia

Product Service

TUV-SUD South Asia

Bangalore, India

Bangalore, India

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

===================================================================== Calibration Table =====================================================================

--------------------------------------------------------------------- General Calibration Setting --------------------------------------------------------------------- Calib. Data Modified : Friday, July 31, 2015 3:06:29 PM Rel. Reference Window : 2.000 % Abs. Reference Window : 0.000 min Rel. Non-ref. Window : 2.000 % Abs. Non-ref. Window : 0.000 min Uncalibrated Peaks : not reported Partial Calibration : Yes, identified peaks are recalibrated Correct All Ret. Times: No, only for identified peaks Average Response : Average all calibrations Average Retention Time: Floating Average New 75% Calibration Report Options : Printout of recalibrations within a sequence: Calibration Table after Recalibration Normal Report after Recalibration If the sequence is done with bracketing: Results of first cycle (ending previous bracket) Default Sample ISTD Information (if not set in sample table): ISTD ISTD Amount Name # [μM] ----|-------------|------------------------- 1 25.00000 Norvaline --------------------------------------------------------------------- --------------------------------------------------------------------- Signal Details --------------------------------------------------------------------- Signal 1: DAD1 A, Sig=260,4 Ref=off --------------------------------------------------------------------- Curve Type : Linear Origin : Included Weight : Equal Recalibration Settings:

--------------------------------------------------------------------- Overview Table ---------------------------------------------------------------------

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 1 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

RT Sig Lvl Amount Area Rsp.Factor Ref ISTD # Compound [μM] -------|-|--|----------|----------|----------|---|---|-|------------------- 2.305 1 1 1.00000 3.83369 2.60845e-1 No No 1 Histidine 2 5.00000 18.27019 2.73670e-1 3 12.50000 48.87408 2.55759e-1 4 25.00000 89.89214 2.78111e-1 5 50.00000 165.02823 3.02978e-1 3.078 1 1 1.00000 4.53686 2.20417e-1 No No 1 Taurine 2 5.00000 18.86520 2.65038e-1 3 12.50000 49.43348 2.52865e-1 4 25.00000 89.88591 2.78130e-1 5 50.00000 165.13036 3.02791e-1 3.361 1 1 1.00000 4.06144 2.46218e-1 No No 1 Serine 2 5.00000 17.19187 2.90835e-1 3 12.50000 48.27116 2.58954e-1 4 25.00000 82.99934 3.01207e-1 5 50.00000 141.53548 3.53268e-1 3.521 1 1 1.00000 3.80690 2.62681e-1 No No 1 Argenine 2 5.00000 17.48554 2.85951e-1 3 12.50000 48.23867 2.59128e-1 4 25.00000 87.86489 2.84528e-1 5 50.00000 155.13309 3.22304e-1 3.656 1 1 1.00000 4.47409 2.23509e-1 No No 1 Glycine 2 5.00000 18.20728 2.74615e-1 3 12.50000 49.33838 2.53352e-1 4 25.00000 88.60124 2.82163e-1 5 50.00000 157.54478 3.17370e-1 3.972 1 1 1.00000 3.63438 2.75150e-1 No No 1 Aspartic acid 2 5.00000 16.24575 3.07773e-1 3 12.50000 47.26416 2.64471e-1 4 25.00000 73.12080 3.41900e-1 5 50.00000 122.87965 4.06902e-1 4.447 1 1 1.00000 4.91760 2.03351e-1 No No 1 Glutamic acid 2 5.00000 17.22200 2.90326e-1 3 12.50000 46.99228 2.66001e-1 4 25.00000 73.68137 3.39299e-1 5 50.00000 123.69010 4.04236e-1 4.859 1 1 1.00000 4.04286 2.47350e-1 No No 1 Threonine 2 5.00000 17.99711 2.77822e-1 3 12.50000 49.70839 2.51467e-1 4 25.00000 84.96769 2.94229e-1 5 50.00000 148.89616 3.35804e-1 5.243 1 1 1.00000 3.99064 2.50587e-1 No No 1 Alanine 2 5.00000 17.64494 2.83367e-1 3 12.50000 50.50707 2.47490e-1 4 25.00000 81.48336 3.06811e-1 5 50.00000 141.07520 3.54421e-1 5.856 1 1 1.00000 4.28069 2.33607e-1 No No 1 Proline 2 5.00000 17.83094 2.80411e-1 3 12.50000 48.49232 2.57773e-1 4 25.00000 80.73975 3.09637e-1 5 50.00000 141.77216 3.52679e-1 6.794 1 1 1.00000 3.65321 2.73732e-1 No No 1 Cystine

2 5.00000 16.65707 3.00173e-1 3 12.50000 42.80335 2.92033e-1 4 25.00000 77.53552 3.22433e-1 5 50.00000 137.41310 3.63866e-1

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 2 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

RT Sig Lvl Amount Area Rsp.Factor Ref ISTD # Compound [μM] -------|-|--|----------|----------|----------|---|---|-|------------------- 6.877 1 1 1.00000 5.84492 1.71089e-1 No No 1 Lysine 2 5.00000 27.45505 1.82116e-1 3 12.50000 81.90633 1.52613e-1 4 25.00000 125.07404 1.99882e-1 5 50.00000 213.28142 2.34432e-1 6.985 1 1 1.00000 3.60393 2.77475e-1 No No 1 Tyrosine 2 5.00000 19.08224 2.62024e-1 3 12.50000 50.68980 2.46598e-1 4 25.00000 95.27022 2.62411e-1 5 50.00000 175.77512 2.84454e-1 7.178 1 1 1.00000 3.94819 2.53281e-1 No No 1 Methionine 2 5.00000 18.35064 2.72470e-1 3 12.50000 50.21463 2.48931e-1 4 25.00000 89.24659 2.80123e-1 5 50.00000 161.04089 3.10480e-1 7.378 1 1 1.00000 5.67687 1.76153e-1 No No 1 Valine 2 5.00000 18.80631 2.65868e-1 3 12.50000 49.32087 2.53442e-1 4 25.00000 81.68585 3.06051e-1 5 50.00000 142.32000 3.51321e-1 7.544 1 1 25.00000 88.56639 2.82274e-1 No Yes 1 Norvaline 2 25.00000 87.19655 2.86709e-1 3 25.00000 98.12215 2.54784e-1 4 25.00000 82.15121 3.04317e-1 5 25.00000 72.62400 3.44239e-1 8.369 1 1 1.00000 3.78179 2.64425e-1 No No 1 Isoleucine 2 5.00000 18.05161 2.76984e-1 3 12.50000 50.89798 2.45589e-1 4 25.00000 86.14241 2.90217e-1 5 50.00000 152.65004 3.27547e-1 8.486 1 1 1.00000 3.74597 2.66954e-1 No No 1 Leucine 2 5.00000 17.92867 2.78883e-1 3 12.50000 50.03949 2.49803e-1 4 25.00000 84.72035 2.95088e-1 5 50.00000 150.09650 3.33119e-1 8.629 1 1 1.00000 3.81862 2.61875e-1 No No 1 Phenyl Alanine --------------------------------------------------------------------- --------------------------------------------------------------------- Peak Sum Table --------------------------------------------------------------------- ***No Entries in table*** --------------------------------------------------------------------- 4 Warnings or Errors : 2 5.00000 19.32251 2.58766e-1 3 12.50000 51.62406 2.42135e-1 4 25.00000 96.05376 2.60271e-1 5 50.00000 177.97539 2.80938e-1

Warning : Overlapping peak time windows at 6.794 min, signal 1 Warning : Overlapping peak time windows at 6.877 min, signal 1 Warning : Overlapping peak time windows at 8.369 min, signal 1 Warning : Overlapping peak time windows at 8.486 min, signal 1 UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 3 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

===================================================================== Calibration Curves =====================================================================

Histidine at exp. RT: 2.305 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99947 Residual Std. Dev.: 0.03194 Formula: y = mx + b m: 1.13672 b: -2.23043e-2 x: Amount y: Area

Area Ratio

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 2.25 0 0.25 0.5 0.75 1 1.25 1.5 1.75 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

5

4

3

2

1

0

1

2

Amount Ratio

Taurine at exp. RT: 3.078 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99947 Residual Std. Dev.: 0.03174 Formula: y = mx + b m: 1.13459 b: -1.73419e-2 x: Amount y: Area

Area Ratio

5

4

3

2

1

0

1

2

Amount Ratio

Serine at exp. RT: 3.361 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99984 Residual Std. Dev.: 0.01523 Formula: y = mx + b

Area Ratio

5

4

m: 9.77128e-1 b: 6.61918e-3 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Argenine at exp. RT: 3.521 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99979 Residual Std. Dev.: 0.01903 Formula: y = mx + b m: 1.07183 b: -1.13097e-2 x: Amount y: Area

Area Ratio

5

4

3

2

1

0

1

2

Amount Ratio

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 4 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Glycine at exp. RT: 3.656 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99981 Residual Std. Dev.: 0.01838 Formula: y = mx + b m: 1.08528 b: -8.16101e-3 x: Amount y: Area

Area Ratio

5

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

4

3

2

1

0

1

2

Amount Ratio

Aspartic acid at exp. RT: 3.972 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99928 Residual Std. Dev.: 0.02756 Formula: y = mx + b

Area Ratio

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

5

4

m: 8.45297e-1 b: 2.16161e-2 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Glutamic acid at exp. RT: 4.447 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99943 Residual Std. Dev.: 0.02454 Formula: y = mx + b

Area Ratio

5

4

m: 8.47014e-1 b: 2.73619e-2 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Threonine at exp. RT: 4.859 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99998 Residual Std. Dev.: 0.00567 Formula: y = mx + b m: 1.02542 b: 1.34798e-3 x: Amount y: Area

Area Ratio

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

5

4

3

2

1

0

1

2

Amount Ratio

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 5 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Alanine at exp. RT: 5.243 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99988 Residual Std. Dev.: 0.01313 Formula: y = mx + b

Area Ratio

5

0 0.25 0.5 0.75 1 1.25 1.5 1.75

4

m: 9.70298e-1 b: 1.12754e-2 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Proline at exp. RT: 5.856 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99999 Residual Std. Dev.: 0.00423 Formula: y = mx + b

Area Ratio

5

0 0.25 0.5 0.75 1 1.25 1.5 1.75 0 0.25 0.5 0.75 1 1.25 1.5 1.75

4

m: 9.73514e-1 b: 6.84062e-3 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Cystine at exp. RT: 6.794 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99978 Residual Std. Dev.: 0.01720 Formula: y = mx + b

Area Ratio

5

4

m: 9.46375e-1 b: -5.83493e-3 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Lysine at exp. RT: 6.877 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99938 Residual Std. Dev.: 0.04455 Formula: y = mx + b m: 1.46663 b: 3.16136e-2 x: Amount y: Area

Area Ratio

5

2.5

2

4

1.5

3

1

2

0.5

1

0

0

1

2

Amount Ratio

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 6 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Tyrosine at exp. RT: 6.985 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99929 Residual Std. Dev.: 0.03922 Formula: y = mx + b m: 1.21227 b: -2.96217e-2 x: Amount y: Area

Area Ratio

5

2

1.5

4

1

3

0.5

2

1

0

0

1

2

Amount Ratio

Methionine at exp. RT: 7.178 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99980 Residual Std. Dev.: 0.01923 Formula: y = mx + b m: 1.10923 b: -1.29841e-2 x: Amount y: Area

Area Ratio

5

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

4

3

2

1

0

1

2

Amount Ratio

Valine at exp. RT: 7.378 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99993 Residual Std. Dev.: 0.00999 Formula: y = mx + b

Area Ratio

5

0 0.25 0.5 0.75 1 1.25 1.5 1.75

4

m: 9.73430e-1 b: 1.59689e-2 x: Amount y: Area

3

2

1

0

1

2

Amount Ratio

Norvaline at exp. RT: 7.544 DAD1 A, Sig=260,4 Ref=off Correlation: 1.00000 Residual Std. Dev.: 0.00000 Formula: y = mx + b

Area Ratio

1

12345

0.8

0.6

m: 1.00000 b: 0.00000 x: Amount y: Area

0.4

0.2

0

0

0.5

1

Amount Ratio

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 7 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Isoleucine at exp. RT: 8.369 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99999 Residual Std. Dev.: 0.00312 Formula: y = mx + b m: 1.05126 b: -2.12938e-3 x: Amount y: Area

Area Ratio

0 0.25 0.5 0.75 1 1.25 1.5 1.75 2 0 0.25 0.5 0.75 1 1.25 1.5 1.75 2

5

4

3

2

1

0

1

2

Amount Ratio

Leucine at exp. RT: 8.486 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99999 Residual Std. Dev.: 0.00310 Formula: y = mx + b m: 1.03336 b: -1.47260e-3 x: Amount y: Area

Area Ratio

5

4

3

2

1

0

1

2

Amount Ratio

Phenyl Alanine at exp. RT: 8.629 DAD1 A, Sig=260,4 Ref=off Correlation: 0.99929 Residual Std. Dev.: 0.03965 Formula: y = mx + b m: 1.22631 b: -2.92805e-2 x: Amount y: Area

Area Ratio

5

2

1.5

4

1

3

0.5

2

1

0

0

1

2

Amount Ratio

=====================================================================

UPLC-DAD 7/31/2015 5:54:40 PM Vikas Kanase

Page 8 of 8

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Sample Name: Amino acids Mix-5uM

===================================================================== Acq. Operator : Seq. Line : 6 Acq. Instrument : UPLC-DAD Location : P1-D-03 Injection Date : 7/6/2015 10:06:50 PM Inj : 1 Inj Volume : 1.000 μl Acq. Method : D:\UPLC\DATA\COE\2015\JULY2015\04AMINOACIDS-VAL-2\04AMINOACIDS-VAL2 2015-07 -06 20-53-07\V-AMINOACIDS-1.M Last changed : 7/6/2015 8:53:08 PM Analysis Method : D:\UPLC\DATA\COE\2015\JULY2015\AA-VAL1\V-AMINOACIDS-1.M (Sequence Method) Last changed : 8/1/2015 2:35:37 PM (modified after loading) Additional Info : Peak(s) manually integrated

DAD1 A, Sig=260,4 Ref=off (D:\UPLC\DATA\COE\2015\JULY2015\AA-VAL1\_006_AMINO ACIDS MIX-5UM-SS1.D)

mAU

35

30

7.525

25

20

15

Area: 16.6504 6.796 Area: 27.5474 6.878 Area: 19.1716 6.988 7.182 7.361

10

8.632

8.372

8.489

Area: 18.0406 5.860

5

5.248

4.863

4.450

3.475

3.604

3.937

3.298

3.030

2.295

0

2

4

6

8

10

min

===================================================================== Internal Standard Report with Performance ===================================================================== Calib. Data Modified : Saturday, August 01, 20152:35:11 PM Multiplier : 1.0000 Dilution : 1.0000 Do not use Multiplier & Dilution Factor with ISTDs

Signal 1: DAD1 A, Sig=260,4 Ref=off

RetTime k' Sig Amount Symm. Width Plates Resol Name [min] [μM] [min] ution -------|------|---|----------||-----|-------|-------|-----|---------- 2.295 - 1 0.00000 0.79 0.1067 2564 - Histidine 3.030 - 1 0.00000 0.71 0.0858 6894 4.49 Taurine 3.298 - 1 0.00000 0.76 0.0779 9937 1.92 Serine 3.475 - 1 0.00000 0.83 0.0702 13563 1.40 Argenine 3.604 - 1 0.00000 0.75 0.0793 11437 1.01 Glycine

Page 1 of 2

UPLC-DAD 8/1/2015 2:35:40 PM

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Sample Name: Amino acids Mix-5uM

RetTime k' Sig Amount Symm. Width Plates Resol Name [min] [μM] [min] ution -------|------|---|----------||-----|-------|-------|-----|---------- 3.937 - 1 0.00000 0.75 0.0750 15247 2.54 Aspartic acid 4.450 - 1 0.00000 0.81 0.0683 23472 4.21 Glutamic acid 4.863 - 1 0.00000 0.83 0.0658 30207 3.61 Threonine 5.248 - 1 0.00000 0.87 0.0633 38038 3.50 Alanine 5.860 - 1 0.00000 0.77 0.0633 47449 5.68 Proline 6.796 - 1 0.00000 0.99 0.0529 91434 9.47 Cystine 6.878 - 1 0.00000 0.95 0.0515 98979 0.92 Lysine 6.988 - 1 0.00000 0.92 0.0569 83589 1.19 Tyrosine 7.182 - 1 0.00000 0.91 0.0586 83280 1.98 Methionine 7.361 - 1 0.00000 0.92 0.0562 95080 1.82 Valine 7.525 - 1 0.00000 0.91 0.0580 93326 1.70 Norvaline 8.372 - 1 0.00000 0.96 0.0605 106235 8.40 Isoleucine 8.489 - 1 0.00000 0.97 0.0608 107852 1.13 Leucine 8.632 - 1 0.00000 0.90 0.0647 98771 1.34 Phenyl Alanine

===================================================================== *** End of Report ***

Page 2 of 2

UPLC-DAD 8/1/2015 2:35:40 PM

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Sample Name: Amino acids Mix-5uM

===================================================================== Acq. Operator : Seq. Line : 6 Acq. Instrument : UPLC-DAD Location : P1-D-03 Injection Date : 7/6/2015 10:06:50 PM Inj : 1 Inj Volume : 1.000 μl Acq. Method : D:\UPLC\DATA\COE\2015\JULY2015\04AMINOACIDS-VAL-2\04AMINOACIDS-VAL2 2015-07 -06 20-53-07\V-AMINOACIDS-1.M Last changed : 7/6/2015 8:53:08 PM Analysis Method : D:\UPLC\DATA\COE\2015\JULY2015\AA-VAL1\V-AMINOACIDS-1.M (Sequence Method) Last changed : 8/1/2015 2:39:21 PM (modified after loading) Additional Info : Peak(s) manually integrated

DAD1 A, Sig=260,4 Ref=off (D:\UPLC\DATA\COE\2015\JULY2015\AA-VAL1\_006_AMINO ACIDS MIX-5UM-SS1.D)

mAU

35

30

7.525

25

20

15

Area: 16.6504 6.796 Area: 27.5474 6.878 Area: 19.1716 6.988 7.182 7.361

10

8.632

8.372

8.489

Area: 18.0406 5.860

5

5.248

4.863

4.450

3.475

3.604

3.937

3.298

3.030

2.295

0

2

4

6

8

10

min

===================================================================== Internal Standard Report with Performance and Noise ===================================================================== Calib. Data Modified : Saturday, August 01, 20152:35:11 PM Multiplier : 1.0000 Dilution : 1.0000 Do not use Multiplier & Dilution Factor with ISTDs

Signal 1: DAD1 A, Sig=260,4 Ref=off

Noise determination:

Time range Noise Noise Noise from | to | (6*SD) | (PtoP) | (ASTM) | Wander | Drift [min] | [min] | [mAU] | [mAU] | [mAU] | [mAU] | [mAU/h] -------|-------|---------|---------|---------|---------|--------- 4.100 4.250 6.336e-2 3.486e-2 - - 36.185 6.000 6.500 0.1294 8.940e-2 - - 45.421 7.700 8.200 0.1290 8.685e-2 - - 31.809

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UPLC-DAD 8/1/2015 2:39:24 PM

Amino-03 (February 2016) FOR ERP USE ONLY DO NOT DISTRIBUTE

Sample Name: Amino acids Mix-5uM

RetTime k' Sig Amount Symm. Width Plates Signal Name [min] [μM] [min] /Noise -------|------|---|----------||-----|-------|-------|------|--------- 2.295 - 1 0.00000 0.79 0.1067 2564 43.7 Histidine 3.030 - 1 0.00000 0.71 0.0858 6894 56.7 Taurine 3.298 - 1 0.00000 0.76 0.0779 9937 56.7 Serine 3.475 - 1 0.00000 0.83 0.0702 13563 63.6 Argenine 3.604 - 1 0.00000 0.75 0.0793 11437 58.6 Glycine 3.937 - 1 0.00000 0.75 0.0750 15247 55.9 Aspartic acid 4.450 - 1 0.00000 0.81 0.0683 23472 63.9 Glutamic acid 4.863 - 1 0.00000 0.83 0.0658 30207 71.8 Threonine 5.248 - 1 0.00000 0.87 0.0633 38038 35.0 Alanine 5.860 - 1 0.00000 0.77 0.0633 47449 34.7 Proline 6.796 - 1 0.00000 0.99 0.0529 91434 40.3 Cystine 6.878 - 1 0.00000 0.95 0.0515 98979 67.7 Lysine 6.988 - 1 0.00000 0.92 0.0569 83589 42.4 Tyrosine 7.182 - 1 0.00000 0.91 0.0586 83280 40.4 Methionine 7.361 - 1 0.00000 0.92 0.0562 95080 42.7 Valine 7.525 - 1 0.00000 0.91 0.0580 93326 190.9 Norvaline 8.372 - 1 0.00000 0.96 0.0605 106235 38.2 Isoleucine 8.489 - 1 0.00000 0.97 0.0608 107852 36.8 Leucine 8.632 - 1 0.00000 0.90 0.0647 98771 37.6 Phenyl Alanine

===================================================================== *** End of Report ***

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UPLC-DAD 8/1/2015 2:39:24 PM