Supplementary Information (SI)

Production and stability study of a hospital parenteral nutrition mixtures for neonates

Authors: Anne-Laure Yailiana, Céline Serreb, Justine Fayardb, Marina Fauconc, Patrick Thomaréc,

Samira Filalib, Christine Pivota, Fabrice Pirota, b*, Emmanuelle Olivierc

1Service Pharmaceutique, Plateforme Fripharm, Groupe Hospitalier Centre Edouard Herriot,

Hospices Civils de Lyon, 5, Place d'Arsonval, F-69437 Lyon cedex 03, France.

2Laboratoire de Recherche et Développement de Pharmacie Galénique Industrielle, UMR 5305,

Plateforme Fripharm, Faculté de Pharmacie, Université Claude Bernard Lyon 1, 8, avenue

Rockefeller, F-69373 Lyon Cedex 08, France.

3Service Pharmaceutique, Site Hôtel-Dieu, Centre Hospitalo-Universitaire de Nantes, 1, place Alexis

Ricordeau, F-44093 Nantes Cedex 01, France.

Methods

Specificity

Interference from the sample excipient (glucose), forced degradation studies and the separation of

amino acids were performed to evaluate the specificity. The glucose concentration was accorded that

one in parenteral nutrition solutions after 1:50 (v:v) dilution. A study demonstrated the influence of

glucose concentration on derivatization of amino acids and their detection [35]. For testing this

influence in hour conditions, it was made firstly a solution of eighteen amino acids at 125 µM;

secondly a mixture of solution of eighteen amino acids at 125 µM with and glucose at 3.4 g/L. The

method specificity was performed by comparing retention time and area under the curve on the two

chromatograms. Furthermore, in order to detect the degradation products, solution prepared was

subjected to forced degradation (NaOH 10 M to obtained pH 8-9, 92°C for 18 hours), the superposition

of these two chromatograms (retention time identical) will be used to show the absence of

interference. The separation of amino acids was study with calibration curve.

Linearity

The theoretical range of calibration was calculated according concentration of each amino acid in the

parenteral nutrition solution. Three ranges of calibration at theoretical concentrations range of 20 and

1

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

1

1000 µM (Table S2) were prepared on account of a range day per operator. The calibration curve was

obtained by linear least square regression analysis. The standard curves were evaluated for intra-day

and inter-day linearity.

Accuracy

A study of accuracy was conducted in order to define the accordance between results obtained and

true value. The study was showed over 3 days on five or seven levels of concentration and theoretical

and measured concentrations were compared. The standard deviation (SD) of individual

measurements was also determined. Recovery should be within the range 100 ± 5%. Accuracy was

expressed as a percentage recovery of theoretical concentration and should not exceed 5%.

Precision

Repeatability (intra-day) was obtained by measuring six replicates of sample preparation. Intermediate

fidelity (inter-day) was verified by comparing the results corresponding to the peak area and retention

time obtained by analyzing six samples.

Limits of detection and quantification

The limit of detection (LOD) and the limit of quantification (LOQ) were determined by the approach of

the standard deviation of the response and the slope of the calibration curve according to ICH

guidelines. The standard deviation was estimated from the intercept of the regression line. The LOD

and the LOQ were determined:

LOD = 3.3 x σ/S and LOQ = 10 x σ/S

where σ was the standard deviation of the intercept of calibration ranges, S was average slope

calibration ranges.

2

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

2

Table of figures for this supplementary section

Fig. S1. Reaction of amino acids derivatization with OPA/3-MPA (A) and FMOC (B).

Fig. S2. Chromatograms of amino acids obtained with condition A by high performance liquid

chromatographic (HPLC) (A) and with condition B by HPLC (B).

Fig. S3. Chromatograms of amino acids obtained by high performance liquid chromatographic (HPLC)

(A) and after forced degradation obtained by HPLC (B).

Fig. S4. Chromatograms of amino acids contained in nutrition parenteral solutions.

3

52

53

54

55

56

57

58

59

60

61

62

3

Figure S1

pH 1020°C60 s

++

3-MPAPrimary amino group OPA derivativeOPA

pH 1020°C60 s

+

FMOC derivativeSecondary amino groupFMOC

A

B

4

63

64

65

66

67

68

69

70

71

72

73

74

4

Figure S2

338 nm

262 nm

262 nm

338 nm

A

B

5

75

76

77

78

5

Figure S3

338 nm

262 nm

262 nm

338 nmA

B

6

79

80

81

82

83

6

Figure S4

338 nm

min

minut

LYS PRO

262 nm

LYS

LEU

ORNILE

PHETRP

MET

VALCYSTINE

TYRTAU

ALA

ARG

THR

GLY

HISSER

GLU

ASP

7

84

85

7

Table S1: Weighing of each amino acid to prepare the stock solution (100 mL HCL 0.1 M) and

standard solutions for chromatographic analysis.

Amino acids Mass (mg)

L-isoleucine 13.12L-leucine 13.12L-valine 11.72L-lysine 14.62L-arginine 17.43L-alanine 8.91L- aspartic acid 13.32L-glutamic acid 14.72L-glycine 7.51L-taurine 5.01L-methionine 5.97L-phenylalanine 6.61L-threonine 4.77L-tryptophan 8.17L-histidine 6.21L-cystine 9.62L-proline 4.61L-serine 4.21L-tyrosine 7.25L-ornithine 5.29

8

86

87

88

8

Table S2: Ranges of concentration for the standard solutions to establish the calibration curve.

Amino acids Range of concentration (µM)

L-isoleucine 100 – 200 – 400 – 600 – 800

L-leucine 200 – 400 – 600 – 800 – 1000

L-valine 200 – 400 – 600 – 800 – 1000

L-lysine 100 – 400 – 600 – 800 – 1000

L-methionine 40 – 80 – 160 – 240 – 320

L-phenylalanine 80 – 160 – 240 – 320 – 400

L-threonine 80 – 160 – 240 – 320 – 400

L-tryptophan 20 – 40 – 80 – 160 – 240 – 320 – 400

L-arginine 100 – 200 – 400 – 600 – 800

L-histidine 80 – 160 – 240 – 320 – 400

L-alanine 200 – 400 – 600 – 800 – 1000

L-aspartic acid 100 – 200 – 400 – 600 – 800

L-glutamic acid 200 – 400 – 600 – 800 – 1000

L-glycine 100 – 200 – 400 – 600 – 800

L-proline 40 – 80 – 240 – 320 – 400

L-serine 80 – 160 – 240 – 320 – 400

L-tyrosine 20 – 40 – 80 – 160 – 240 – 320 – 400

L-cystine 40 – 80 – 160 – 240 – 320

L-taurine 20 – 40 – 80 – 160 – 240

L-ornithine 80 – 160 – 240 – 320 – 400

9

89

90

9

TO DELETE

Table S3: Development of the amino acid chromatographic assay to obtain optimized chromatographic

conditions.

ParametersMethod

Condition A Condition B

Stationary phase (column) Zorbax Eclipse-AAA 3.5 µm, 150 mm x 4.6 mm

Poroshell HPH-C18 2.7µm, 3.0 mm x 150 mm

Mobile phase Gradient with:Eluent A = phosphate buffer pH 7.82Eluent B = 45% of ACN. 45% of MeOH and 10% ultra-pure water

Gradient with:Eluent A = buffer Na2HPO4 10 mM / Na2B4O7 (10 H2O) 10 mM / NaN3 0.5 mM pH 8.2 with HCl 37%Eluent B = 45% of ACN. 45% of MeOH and 10% ultra-pure water

Flow rate (mL/min) 0.6 0.64

Run time (minutes) 31 min 27 min

Injector temperature ambient ambient

Column temperature (°C) 40 40

Volume of injection loop (µL) 18 12

Detection wavelength (nm) 262 and 338 262 and 338

10

91

92

93

94

95

10

Table S4 S3: System suitability parameters ensuring the validity of the amino acid analytical

procedure in HPLC system.

Amino acids

System suitability parameters

Retention time (min) ± S.D.

Theoretical plate number Asymmetry Resolution

(R)Capacity factor k’

L-isoleucine 14.36 ± 0.08 193745 0.81 1.86 13.27

L-leucine 15.15 ± 0.08 215500 0.80 6.01 14.05

L-valine 12.37 ± 0.06 164518 0.90 4.34 11.28

L-lysine 15.85 ± 0.09 282170 0.81 5.43 14.72

L-methionine 12.67 ± 0.06 215684 1.03 2.53 11.57

L-phenylalanine 14.13 ± 0.07 251097 0.99 3.86 13.04

L-threonine 7.21 ± 0.03 85004 1.03 2.37 6.19

L-tryptophan 13.73 ± 0.06 267520 0.99 9.8 12.62

L-arginine 8.24 ± 0.04 115120 1.07 9.93 7.15

L-histidine 6.64 ± 0.03 94727 1.05 11.24 5.61

L-alanine 8.66 ± 0.05 96194 0.85 4.29 7.60

L-aspartic acid 1.51 ± 0.01 2528 0.42 5.68 0.63

L-glutamic acid 2.42 ± 0.02 4654 0.55 7.8 1.76

L-glycine 6.96 ± 0.04 68571 0.89 3.47 5.94

L-proline 19.73 ± 0.09 68480 0.99 18.37 18.57

L-serine 5.53 ± 0.02 54779 0.99 21.91 4.59

L-tyrosine 10.23 ± 0.05 184945 0.99 14.88 9.13

L-cystine 11.92 ± 0.06 227622 0.91 17.32 10.80

L-taurine 8.79 ± 0.05 173100 0.86 1.69 7.78

L-ornithine 14.89 ± 0.07 308370 0.82 4.37 13.87

11

96

97

98

99

11

Table S5 S4: Influence of glucose concentration on amino acid derivatization.

Amino acidRetention time Peak Area

Without glucose With glucose Without glucose With glucose

L-aspartic acid 1.51 1.50 25.89 22.31

L-glutamic acid 2.42 2.43 36.83 40.21

L-serine 5.53 5.52 37.95 34.14

L-histidine 6.64 6.62 18.75 16.49

L-glycine 6.96 6.94 38.96 34.77

L-threonine 7.21 7.20 42.33 37.37

L-arginine 8.24 8.27 36.75 32.28

L-alanine 8.66 8.65 38.75 34.21

L-tyrosine 10.23 10.25 37.16 32.48

L-cystine 11.92 11.90 90.38 80.45

L-valine 12.37 12.35 40.06 35.80

L-methionine 12.67 12.70 45.44 40.00

L-tryptophan 13.73 13.75 34.95 30.75

L-phenylalanine 14.13 14.12 37.36 33.13

L-isoleucine 14.36 14.33 36.11 31.97

L-leucine 15.15 15.12 85.46 76.36

L-lysine 15.85 15.87 5.92 6.21

L-proline 19.73 19.75 69.30 65.21

12

100

101

102

12

Table S6 S5: Validation parameters of the amino acid assay by HPLC. The repeatability and fidelity data are the mean ± SD of n = 18 experimental determinations. CV: coefficient of variation.

Amino acids

Regression analysis Repeatability Fidelity LOD LOQ Capability

Linearity range (µM) Curve’s equation r Conc.

(µM)CV(%)

Conc.(µM)

CV(%) (µM) (µM) Capability

valueRisk level

L-isoleucine 100 - 800 y = 0.54x + 1.19 0.997 403.07 ± 14.53 3.61 396.81 ± 6.52 1.64 10.69 32.38 15.00 < 0.01%L-leucine 200 - 1000 y = 0.54x + 0.87 0.997 989.91 ± 31.95 3.23 394.11 ± 4.96 1.26 13.42 40.61 17.22 < 0.01%L-valine 200 - 1000 y = 0.54x + 0.58 0.994 404.00 ± 14.82 3.67 406.55 ± 3.85 0.95 13.01 39.41 17.08 < 0.01%L-lysine 100 - 1000 y = 0.88x - 14.76 0.998 404.00 ± 14.82 3.67 387.22 ± 8.55 2.21 20.35 61.68 14.44 < 0.01%L-methionine 40 - 320 y = 0.54x - 0.05 0.997 159.27 ± 5.59 3.51 156.77 ± 3.44 2.20 3.91 11.85 15.00 < 0.01%L-phenylalanine 80 - 400 y = 0.50x - 1.26 0.998 159.30 ± 7.43 4.66 156.76 ± 3.90 2.49 7.74 23.45 17.22 < 0.01%L-threonine 80 - 400 y = 0.54x - 2.21 0.999 397.49 ± 3.45 0.87 156.76 ± 3.90 2.49 5.12 15.51 15.56 < 0.01%L-tryptophan 20 - 400 y = 0.42x - 0.81 0.996 157.30 ± 4.62 2.93 153.26 ± 2.87 1.87 5.14 15.58 13.06 < 0.01%L-arginine 100 - 800 y = 0.55x + 1.91 0.996 406.99 ± 16.27 4.00 404.03 ± 5.87 1.45 11.98 36.29 16.00 < 0.01%L-histidine 80 - 400 y = 0.38x - 1.94 0.998 157.99 ± 5.96 3.77 156.82 ± 2.63 1.68 9.70 29.40 14.17 < 0.01%L-alanine 200 - 1000 y = 0.54x + 2.50 0.995 1009.70 ± 46.23 4.58 403.14 ± 9.09 2.26 14.15 42.88 13.67 < 0.01%L-aspartic acid 100 - 800 y = 0.51x + 5.49 0.998 407.16 ± 12.65 3.11 397.32 ± 5.83 1.47 8.74 26.48 14.00 < 0.01%L-glutamic acid 200 - 1000 y = 0.54x + 1.85 0.996 404.89 ± 12.94 3.20 398.65 ± 5.97 1.50 14.03 42.52 13.75 < 0.01%L-glycine 100 - 800 y = 0.52x + 1.05 0.997 407.33 ± 14.87 3.65 400.11 ± 7.69 1.92 6.12 18.55 15.42 < 0.01%L-proline 40 - 400 y = 0.46x + 3.66 0.991 166.39 ± 4.99 3.00 159.85 ± 2.29 1.44 9.51 28.83 14.63 < 0.01%L-serine 80 - 400 y = 0.54x + 0.56 0.995 398.06 ± 18.17 4.56 159.12 ± 3.34 2.10 5.24 15.87 16.67 < 0.01%L-tyrosine 20 - 400 y = 0.50x + 1.00 0.997 20.26 ± 0.79 3.92 158.19 ± 2.69 1.70 2.80 8.47 13.06 < 0.01%L-cystine 40 - 320 y = 0.89x + 2.23 0.999 19.07 ± 0.63 3.29 164.27 ± 1.62 0.99 7.36 22.23 13.33 < 0.01%L-taurine 20 - 240 y = 0.64x – 0.91 0.995 39.74 ± 1.07 2.68 38.58 ± 1.52 3.93 1.72 5.21 15.00 < 0.01%L-ornithine 80 - 400 y = 0.71x – 3.42 0.993 237.03 ± 8.33 3.52 230.86 ± 10.54 4.57 18.59 56.32 15.42 < 0.01%

13

103104

105

13

TO DELETE

Table S7: Accuracy of the amino acid assay by HPLC. Each data is the mean ± Standard

Deviation of 15 experimental determinations.

Amino acids Mean Recovery ± SD Recovery confidence interval

L-isoleucine 0.99 ± 0.04 [ 97% ; 100% ]L-leucine 1.00 ± 0.03 [ 98% ; 101% ]L-valine 0.99 ± 0.04 [ 97% ; 101% ]L-lysine 0.97 ± 0.05 [ 95% ; 100% ]L-methionine 0.99 ± 0.03 [ 97% ; 100% ]L-phenylalanine 0.98 ± 0.03 [ 97% ; 100% ]L-threonine 0.99 ± 0.03 [ 98% ; 100% ]L-tryptophan 1.01 ± 0.06 [ 98% ; 103% ]L-arginine 0.99 ± 0.05 [ 96% ; 101% ]L-histidine 0.98 ± 0.03 [ 97% ; 100% ]L-alanine 1.00 ± 0.04 [ 98% ; 102% ]L-aspartic acid 1.02 ± 0.04 [ 98% ; 100% ]L-glutamic acid 1.01 ± 0.03 [ 98% ; 100% ]L-glycine 1.01 ± 0.04 [ 98% ; 100% ]L-proline 1.01 ± 0.08 [ 98% ; 105% ]L-serine 1.00 ± 0.04 [ 98% ; 102% ]L-tyrosine 1.00 ± 0.06 [ 98% ; 103% ]L-cystine 0.98 ± 0.02 [ 97% ; 99% ]L-taurine 0.99 ± 0.05 [ 97% ; 102% ]L-ornithine 1.03 ± 0.04 [ 101% ; 104% ]

14

106

107

108

109

110

14

TO DELETE

Table S8: Repeatability of the amino acid assay by HPLC. Each data is the mean ± Standard

Deviation of 18 experimental determinations.

Amino acids Mean concentration (µM) ± SD Variation coefficient (%)

L-isoleucine 403.07 ± 14.53 3.61L-leucine 989.91 ± 31.95 3.23L-valine 404.00 ± 14.82 3.67L-lysine 404.00 ± 14.82 3.67L-methionine 159.27 ± 5.59 3.51L-phenylalanine 159.30 ± 7.43 4.66L-threonine 397.49 ± 3.45 0.87L-tryptophan 157.30 ± 4.62 2.93L-arginine 406.99 ± 16.27 4.00L-histidine 157.99 ± 5.96 3.77L-alanine 1009.70 ± 46.23 4.58L-aspartic acid 407.16 ± 12.65 3.11L-glutamic acid 404.89 ± 12.94 3.20L-glycine 407.33 ± 14.87 3.65L-proline 166.39 ± 4.99 3.00L-serine 398.06 ± 18.17 4.56L-tyrosine 20.26 ± 0.79 3.92L-cystine 19.07 ± 0.63 3.29L-taurine 39.74 ± 1.07 2.68L-ornithine 237.03 ± 8.33 3.52

15

111

112

113

114

115

15

TO DELETE

Table S9: Intermediate fidelity of the amino acid assay by HPLC. Each data is the mean ± Standard

Deviation of 18 experimental determinations.

Amino acids Mean concentration (µM) ± SD Variation coefficient (%)

L-isoleucine 396.81 ± 6.52 1.64L-leucine 394.11 ± 4.96 1.26L-valine 406.55 ± 3.85 0.95L-lysine 387.22 ± 8.55 2.21L-methionine 156.77 ± 3.44 2.20L-phenylalanine 156.76 ± 3.90 2.49L-threonine 156.76 ± 3.90 2.49L-tryptophan 153.26 ± 2.87 1.87L-arginine 404.03 ± 5.87 1.45L-histidine 156.82 ± 2.63 1.68L-alanine 403.14 ± 9.09 2.26L-aspartic acid 397.32 ± 5.83 1.47L-glutamic acid 398.65 ± 5.97 1.50L-glycine 400.11 ± 7.69 1.92L-proline 159.85 ± 2.29 1.44L-serine 159.12 ± 3.34 2.10L-tyrosine 158.19 ± 2.69 1.70L-cystine 164.27 ± 1.62 0.99L-taurine 38.58 ± 1.52 3.93L-ornithine 230.86 ± 10.54 4.57

16

116

117

118

119

120

16

TO DELETE

Table S10: Limits of detection and quantification of the amino acid assay by HPLC.

Amino acids LOD (µM) LOQ (µM)

L-isoleucine 10.69 32.38L-leucine 13.42 40.61L-valine 13.01 39.41L-lysine 20.35 61.68L-methionine 3.91 11.85L-phenylalanine 7.74 23.45L-threonine 5.12 15.51L-tryptophan 5.14 15.58L-arginine 11.98 36.29L-histidine 9.70 29.40L-alanine 14.15 42.88L-aspartic acid 8.74 26.48L-glutamic acid 14.03 42.52L-glycine 6.12 18.55L-proline 9.51 28.83L-serine 5.24 15.87L-tyrosine 2.80 8.47L-cystine 7.36 22.23L-taurine 1.72 5.21L-ornithine 18.59 56.32

17

121

122

123

124

17

TO DELETE

Table S11: Capability of the amino acid assay by HPLC.

Amino acids Capability value Estimated risk level

L-isoleucine 15.00 < 0.01%L-leucine 17.22 < 0.01%L-valine 17.08 < 0.01%L-lysine 14.44 < 0.01%L-methionine 15.00 < 0.01%L-phenylalanine 17.22 < 0.01%L-threonine 15.56 < 0.01%L-tryptophan 13.06 < 0.01%L-arginine 16.00 < 0.01%L-histidine 14.17 < 0.01%L-alanine 13.67 < 0.01%L-aspartic acid 14.00 < 0.01%L-glutamic acid 13.75 < 0.01%L-glycine 15.42 < 0.01%L-proline 14.63 < 0.01%L-serine 16.67 < 0.01%L-tyrosine 13.06 < 0.01%L-cystine 13.33 < 0.01%L-taurine 15.00 < 0.01%L-ornithine 15.42 < 0.01%

18

125

126

127

128

18