Review Team (February 2016)

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