2017.03 Amino Acids (Tryptophan) - Amino-02
2017.03 (JULY 2018) Amino-02 TRP_AOAC_2017.03_MLT_Corrected 080218
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HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis
AOAC First Action 2017.03 Multi Laboratory Study
Final Report
July 20, 2018
Study Director:
Jonathan Draher
Abbott Nutrition, Columbus Ohio USA
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Study Information
Study Title: Collaborative Study of AOAC First Action 2017.03 for the HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis.
Study Objective: The objective of this study is to complete validation of AOAC First Action 2017.03 for the quantification of tryptophan in infant, pediatric, and adult nutritionals by determining method reproducibility and repeatability. Study Director
Jonathan Draher Abbott Nutrition 3300 Stelzer Road Columbus, OH 43219 P: 614 624-4231 jonathan.draher@abbott.com
Study Monitors: AOAC International (SPIFAN) Official Method Board or delegated personnel.
MLT Overview
Participating laboratories were asked to set up the method (Appendix A), analyze two practice samples in duplicate, and report the results to the study directors. After successful analysis of the practice samples, study participants were asked to analyze 14 SPIFAN matrices in duplicate. The samples were split into groups of 14 and 14 per the data reporting templates, and each group was analyzed on a separate day. Participants were asked to reconstitute all powder samples by diluting either 10 or 25 grams of powder to 100 or 225 grams respectively with water. Electronic excel templates for reporting sample weights and results were provided. After completion of the study, the data were analyzed using the AOAC International Interlaboratory Study Workbook for Blind (unpaired) Replicates, Version 2.1 to calculate repeatability standard deviation, s(r), reproducibility standard deviation, s(R), repeatability relative standard deviation, RSD(r), reproducibility relative standard deviation, RSD(R), and HORRAT value to detect outliers.
Table 1: MLT Sample Matrices with Codes
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Blind Duplicate Codes
Product Description
Batch
K16NTAV E10NWZC
CULF358 TJHR217 KDOX966 SWUO667 DYLB360 FPTE312 EFXN778 NSRB999 XKIP216 KGSZ273 RQXQ518 LYNY751 ECHL425 URTF231
GBZC169 OACN211 ATAN351 MYHK654 ZMQM883 DOMY545 BFAO941 JSDT587 HYJU890 LTCT316 GVPE615 PZGP859 UOPM297 WKHN288
Milk-based Infant Formula Powder Soy-based Infant Formula Powder
410057652Z 410457651Z 00729RF00 00730RF00 4052755861 4044755861
Milk-based Partially Hydrolyzed Infant Formula Powder Soy-based Partially Hydrolyzed Infant Formula Powder Adult Nutritional Ready-to-feed- High Fat Adult Nutritional Ready-to-feed- High Protein Milk-based Child Formula Powder Progress Gold Milk-based Infant Formula Stage 1 Powder Illuma Milk-based Infant Formula Ready-to-feed
EV4H2R CLC10-B
SRM 1849a a
00866RF00 00859RF00 00795RF00 50350017W1
Milk-based Child Formula Powder Adult Low Fat Nutritional Powder Infant Elemental Formula Powder FOS/GOS-based Infant Formula Powder
Table 2: AOAC 2017.03 MLT Participating Laboratories Laboratory Address
Country
Abbott Laboratories
Buildig 14 Caohejing SBP,Phase III, No.1306 Tian Lin Road, Minhang District, Shanghai 200233, P.R. China
China
Abbott Laboratories Laboratoire AQUANAL
3300 Stelzer Rd, Columbus OH 43219 151 bis, avenue Jean Jaures, 33600 PESSAC
USA
France
AsureQuality
131 Boundary Rd, Auckland 1140
New Zealand
Eurofins Nutrition Analysis Center Food Industry Research and Development Institute (FIRDI) Nestle Quality Assurance Center
2200 Rittenhouse St. Suite 150 Des Moines, Iowa 50321
USA
331 Shih-Pin Rd, Hsinchu, 300 Taiwan R.O.C.
Taiwan
6625 Eiterman Rd. Dublin, Oh 43016
USA
NIFC
65 Pham Than Duat Street, Cau Giav district, Hanoi, Vietnam 147 Industrial Park Road Georgia, VT 05468-2109
Vietnam
Perrigo Nutritionals Syngene International Limited
USA India
Block: S-15, Biocon Park, Plot No. 2&3 Bommasandra IV Phase, Jigani Link Road Bangalore 560 100, Karnataka, India
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Results and Discussion Two of the twelve laboratories that signed up to participate withdrew from the study because of time constraints. None of these laboratories attempted to set up the method. The remaining ten laboratories completed the study. Each laboratory reported results for all fourteen matrices used for the study. All tryptophan MLT data are summarized in the tables below.
Table 3: AOAC 2017.03 L-Tryptophan Data (mg/100g RTF) from Analysis of Blind Duplicates, Individual Results
Lab
K16NTAV
E10NWZC
410057652Z
410457651Z
00729RF00
1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 8 9
18.42 19.54 19.28 19.40 20.61 20.13 19.02 20.22 19.00 20.06 68.09 70.29 72.30 72.20 68.89 74.59 65.13 74.44 71.79 88.38 24.73 23.22 23.08 23.11 22.75 24.09 21.74 25.07 22.82 23.05
18.49 20.13 19.32 19.90 18.88 20.53 18.52 20.28 18.57 20.93 68.86 71.88 59.77 67.30 68.30 73.21 66.87 73.91 70.90 69.03 24.95 22.00 23.19 23.28 22.00 24.39 23.76 24.58 22.28 23.55
17.99 18.20 17.60 18.03 16.47 18.89 17.64 18.65 17.56 18.55 24.69 23.50 24.77 25.08 24.53 25.53 23.73 26.70 23.98 25.53 21.02 21.58 21.13 21.90 21.12 29.93 21.02 21.94 20.80 21.93
18.01 18.70 17.86 18.59 17.32 18.91 17.24 18.73 17.46 19.37 25.12 23.44 24.72 26.07 23.92 24.51 24.12 26.01 23.67 26.24 21.04 22.15 21.21 21.68 20.95 31.49 20.23 22.08 20.09 22.44
24.70 25.67 24.85 25.20 26.45 26.67 24.44 26.67 23.99 25.92 23.82 24.32 23.91 23.82 24.51 24.71 23.44 25.03 23.08 24.81 31.57 32.62 32.35 32.06 34.43 34.01 31.38 33.60 31.92 33.03
24.81 25.92 24.85 25.39 23.54 26.63 24.18 27.18 23.72 26.57 23.08 23.35 23.95 24.46 22.97 25.01 22.86 25.11 22.79 24.24 32.00 33.45 32.62 33.04 33.02 34.10 30.50 33.56 32.23 34.44
17.97 15.15 17.70 18.26 16.43 21.53 16.95 19.02 17.42 19.86 21.27 22.07 21.70 22.40 20.63 23.34 20.93 22.84 21.12 22.79 22.64 22.63 21.89 23.12 23.60 22.81 21.51 23.77 21.82 23.03
18.26 15.75 17.58 18.31 16.97 19.95 14.77 18.82 17.27 20.51 21.43 22.18 22.03 22.90 21.27 23.03 21.02 22.94 21.01 22.86 22.52 21.92 22.43 23.91 21.92 23.39 21.50 23.47 21.50 22.48
85.57 85.60 89.61 91.00 89.17 94.47 86.03 94.28 91.62 94.05 17.75 18.31 17.96 18.11 18.45 18.66 17.40 18.87 17.61 18.86
86.31 89.29 90.62 93.30 86.76 95.84 85.30 94.69 91.91 94.99 18.13 18.55 17.91 18.23 17.73 18.71 16.89 18.87 17.73 18.03
10
Lab
00730RF00
4052755861
4044755861
EV4H2R
CLC10-B
10
Lab
00866RF00
00859RF00
00795RF00
50350017W1
10
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Table 4: Statistical Analysis of MLT Blind Duplicates Sample ID: K16NTAV
E10NWZC
410057652Z
410457651Z
00729RF00
Total number of laboratories Total number of replicates
10 20
10 20
10 20
10 20
9
18
Overall mean of all data (grand mean), mg/100g Repeatability standard deviation Reproducibility standard deviation Repeatability relative standard deviation, % Reproducibility relative standard deviation, %
19.56
18.09
25.41
17.92
90.52
0.50 0.79
0.33 0.73
0.23 1.08
0.65 1.78
1.22 3.72
2.5 4.1
1.8 4.0
0.9 4.2
3.6 9.9
1.3 4.1
HORRAT value
0.56
0.55
0.61
1.35
0.72
Sample ID:
00730RF00
4052755861
4044755861
EV4H2R
CLC10-B
Total number of laboratories Total number of replicates
8
10 20
10 20
10 20
10 20
16
Overall mean of all data (grand mean), mg/100g Repeatability standard deviation Reproducibility standard deviation Repeatability relative standard deviation, % Reproducibility relative standard deviation, %
70.42
24.79
23.96
21.99
18.14
1.45 2.97
0.44 1.00
0.51 0.79
0.22 0.88
0.29 0.55
2.1 4.2
1.8 4.0
2.1 3.3
1.0 4.0
1.6 3.0
HORRAT value
0.71
0.58
0.47
0.56
0.41
Sample ID:
00866RF00
00859RF00
00795RF00
50350017W1
Total number of laboratories Total number of replicates
10 20
9
10 20
10 20
18
Overall mean of all data (grand mean), mg/100g Repeatability standard deviation Reproducibility standard deviation Repeatability relative standard deviation, % Reproducibility relative standard deviation, %
23.38
21.35
32.80
22.59
0.60 1.02
0.32 0.67
0.58 1.09
0.50 0.80
2.5 4.4
1.5 3.1
1.8 3.3
2.2 3.5
HORRAT value
0.62
0.44
0.50
0.50
Repeatability (RSD r )
For all fourteen of the product matrices analyzed, the overall repeatability averaged 2.1% RSD with a range of 0.9 – 3.6% RSD. The SMPR requirements of ≤ 3% RSD for samples with L-tryptophan levels of 5-50 mg/100g RTF or ≤ 2% RSD for products with levels ranging from 50 - 2500 mg/100g RTF were met for thirteen of the fourteen matrices analyzed. All SPIFAN II product matrices except 00730RF00 and 00729RF00 had L- tryptophan levels between 5-50 mg/100g RTF. The L-tryptophan levels in 00730RF00 and 00729RF00 are in
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the 50-2500 mg/100g RTF range. Three of the matrices had labs that were flagged as outliers using the Cochran’s test and the flagged data was removed for these matrices. Reproducibility (RSD R ) For all product matrices, reproducibility averaged 4.2% RSD with a range of 3.0 - 9.9% RSD. The standard method performance requirements for reproducibility of ≤ 4% RSD for samples with L-tryptophan levels of 5- 50 mg/100g RTF and ≤ 3% RSD for samples with L-tryptophan levels of 50-2500 mg/100g RTF were met for eleven of the fourteen product matrices analyzed. The three product matrices that did not meet the reproducibility requirement were 00729RF00 (Adult Nutritional RTF High Fat), 00730RF00 (Adult Nutritional RTF High Protein), and 410457651Z (Soy-based Partially Hydrolyzed Infant Formula Powder). Reproducibility for 00729RF00 was 4.1% RSD and was slightly above the requirement of ≤ 3% RSD. Reproducibility for 00730RF00 was 4.2% and was also slightly above the requirement of ≤ 3% RSD. The slightly higher reproducibility for these liquid matrices can be attributed to the sample losing homogeneity as it has aged. See figures 1 and 2 for pictures of 00730RF00 and note the large chunks that are easily seen throughout this product. Observations were made from participating labs about the clumping in liquid samples and concerns about this sample’s homogeneity were also noted by the participating laboratories. It should also be noted that the MLT study for AOAC first action method 2015.09 (Vitamin K 1 ) reported similar problems with this sample. Reproducibility for 410457651Z was 9.3% RSD and was above the requirement of ≤ 4% RSD. It should be noted that this sample had an RSDr and RSD iR of 4.95% and 6.92% respectively in the SLV conducted for this method in early 2017. Therefore, it was expected to see a higher reproducibility in this product in the MLT study. Statistical Analysis of MLT Data Using the AOAC International Interlaboratory Study Workbook for Blind (unpaired) Replicates, Version 2.1 to analyze the L-tryptophan MLT data, statistical outliers were identified for three for the fourteen product matrices. The data identified as outliers was removed from the data pool. Data for 410057652Z, 00730RF00, and 00859RF00 were flagged as Cochran outliers. HORRAT values ranged from 0.41 – 1.35 for the fourteen product matrices analyzed. Thirteen of the fourteen product matrices had HORRAT values below 1.00. General Comments There were no major deviations to the MLT protocol during the MLT study. One laboratory did not analyze the practice samples. One laboratory had to re-test day 2 of the study after an error in the preparation of the enzyme was discovered. Once the error was corrected the lab reported acceptable results for day 2. A mistake was made in reporting results for Lab 6. They inadvertently switched samples 00729RF00 and 00730RF00 on day 2 of testing and reported the incorrect results for these two samples. The error was caught and the correct data is reported in the final study report. Analytical columns from multiple vendors were used by the participating labs to complete this MLT study. No significant impact on chromatography was shown using these different column brands.
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Figure 1. Photos of SPIFAN II Product 00730RF00 (Adult Nutritional High Protein RTF).
Figure 2. Photo of clumping in SPIFAN II Product 00730RF00 (Adult Nutritional High Protein RTF).
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Appendix A
This method is applicable to the determination of total tryptophan in infant formula and adult/pediatric nutritional formulas. A sample amount targeting about 30 – 50 mg of protein is added to the sample preparation. The limit of quantitation of a typical ready to feed formula is approximately 0.18 mg/100g. A. SAFETY ALL REAGENTS AND EQUIPMENT SHOULD BE HANDLED IN A MANNER CONSISTENT WITH SAFE LABORATORY PRACTICES. REAGENTS AND SAMPLES SHOULD BE TREATED WITH THE RESPECT AND CARE THAT ANY CHEMICAL REQUIRES. REAGENTS WHICH ARE ALLOWED TO COME IN CONTACT WITH THE SKIN, EYES, OR CLOTHING SHOULD BE FLUSHED IMMEDIATELY WITH LARGE VOLUMES OF WATER. IMMEDIATE MEDICAL ATTENTION SHOULD BE OBTAINED IF ANY REAGENT IS INGESTED OR ALLOWED TO COME IN CONTACT WITH THE EYES. ALL OF THE SOLVENTS USED IN THIS METHOD ARE HIGHLY FLAMMABLE AND SHOULD BE KEPT AWAY FROM FLAMES AND OTHER IGNITION SOURCES. METHANOL IS A TOXIC CUMULATIVE POISON THAT CAN CAUSE BLINDNESS. PHOSPHORIC ACID AND HYDROCHLORIC ACID MAY CAUSE IRRITATION TO SKIN AND EYES IF DIRECT CONTACT IS MADE. USE CARE WHEN HANDLING STREPTOMYCES GRISEUS ENZYME: MAY BE HARMFUL IF INHALED. MAY CAUSE RESPIRATORY TRACT, EYE AND SKIN IRRITATION. MAY CAUSE ALLERGIC RESPIRATORY AND SKIN REACTION .
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B. PRINCIPLE Tryptophan is released (hydrolyzed) from intact protein using a combination of proteolytic enzymes found in pronase, isolated from Streptomyces griseus, for this purpose. The pronase enzyme powder contains at least 10 proteolytic enzymes (depending on the source, supplier etc.), which hydrolyze peptide bonds internally (endoproteases) and externally (exopeptidases), either at the N-terminal end (amino peptidases) or at the carboxy terminus (carboxypeptidases). The protein is thus "attacked" on different sites simultaneously releasing tryptophan in a relatively short period of time. Following proteolysis, free tryptophan is quantitated by reverse phase, isocratic HPLC and fluorescence detection, which provide for a selective and specific determination of tryptophan in nutritional products. The enzymes in pronase self-digest to produce background tryptophan in the absence of sample. Consequently, the enzyme system is non-specific for the sample tryptophan, and a blank subtraction is mandatory. Using this approach, recoveries of free tryptophan spikes as well as tryptophan from BSA spikes are found essentially quantitative, indicating near comparable self- digestion rates with and without sample. Sample preparation consists of adding a weighed sample, the enzyme solution, internal standard (5-methyl-DL-tryptophan) and Trizma buffer into a tube. A small amount of methanol is added as a bactericidal agent. The preparation is mixed and incubated at 50°C for sixteen hours (overnight), to assure complete hydrolysis of all sample types. (While many samples are fully hydrolyzed within 6 hours, some have been found to require longer, thus 16 hours minimum for full applicability). After hydrolysis, the sample-enzyme mixture is diluted to 50 mL with methanol/water and filtered. The sample is injected onto a C-8 column with reference standards and enzyme blank preparations and the analytes of interest detected and quantified fluorometrically. C. APPARATUS 1. Equipment a) Analytical Column: YMC PAC C8 50 × 3 mm column with 3 micron particle size – Part #OC12S03-053030 – or equivalent. b) HPLC system with autosampler, column oven to maintain 30°C, Fluorescence Detector, binary or quaternary HPLC Pump with degasser, HPLC peak area integrator or chromatography data processor. c) Constant temperature oven capable of maintaining 50 (+/- 1) degree Centigrade 2. Instrument Parameters and Conditions a) Mobile Phase Flow Rate : 0.5 mL /minute b) Column Temperature: 30 degrees Centigrade c) Injection volume: 10 µ L. d) Detector settings: Excitation wavelength to 295 nm and emission wavelength to 345 nm
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e) Typical PMT setting: 7 for Agilent G1321A (see F.3.a) f) Run time: 8.5 minutes, or as needed to fully collect the 5-methyl-DL-tryptophan peak.
D. REAGENTS 1. Standards
L-Tryptophan USP Standard Cat #1700501, or equivalent. 2. Chemicals a) Methanol, HPLC grade b) Sodium Phosphate Monobasic, HPLC grade. c) Phosphoric Acid, (85%) d) Trizma Base e) Hydrochloric Acid, 6N.
f) Laboratory Water – minimum of 18 mega ohm conductivity g) 5-Methyl-DL-tryptophan Sigma Cat # M-0534, or equivalent. h) Protease from Streptomyces griseus : Sigma Cat # P-5147, or equivalent. 3. Solutions a) Hydrochloric Acid, 1.0N Solution. b) Trizma Buffer, 0.1M, pH 8.5. c) Protease Solution - Weigh 0.125 ± 0.010 grams to 25 mL with Trizma buffer. Scale volume as needed with proportional weight. Expiration: 6 hours. d) 5-Methyl-DL-tryptophan, Internal Standard Solution - 0.0400 ± 0.0020 grams to 50 mL with water. Initially add ~25 mL water and stir with small stir bar. Due to slow dissolution, may stir overnight in a refrigerator. Store frozen. Expiration: 6 months e) Tryptophan Stock Standard Solution - (0.0100 ± 0.0010 grams per 100 mL) Store frozen. Expiration: 6 months. h) Working Standard Solutions: Combine the appropriate aliquot of tryptophan and internal standard stock solutions in separate 50 mL volumetric flasks for four working levels. Standard TRP Stock Sol. Int. Std. Stock VERY LOW 0.20 mL 0.5 mL LOW 1.0 mL 0.5 mL MID 5.0 mL 0.5 mL HIGH 10.0 mL 0.5 mL
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Add 12.0 mL of methanol to each of the four flasks containing the standard/internal standard mixtures, and bring to volume with water. Transfer diluted standards to vials for frozen storage. Expiration: 6 months. g) Mobile Phase: (18:82, Methanol/0.05 M Phosphate Buffer, pH 2.3) h) Column Rinse Solution: (25% methanol in water)
E. PROCEDURE
1. Sample Preparation
a) Set heating unit to 50°C prior to experiment. b) Using an analytical balance, tare a 50 mL centrifuge tube.
c) Weigh liquid samples and reconstituted powders directly into tarred centrifuge tubes using a disposable transfer pipette. For non-reconstituted powder samples weigh into a tarred tube with a spatula. Record weights. d) Add 0.5 mL (500 µ L) of protease enzyme solution to all samples. e) Prepare no fewer than two "blank" enzyme solutions per run. Blank solutions will contain enzyme, internal standard, and buffer only. Otherwise follow sample preparation. f) Add 0.5 mL of 5-methyl-DL-tryptophan internal standard to all tubes including the enzyme blanks. g) Add 3.0 mL of 0.1M, pH 8.5 Trizma buffer and 200 µ L methanol to all tubes including enzyme blanks. Cap tubes and vortex lightly (about 2 seconds) to mix contents. Be sure powder samples are completely dissolved but do not over agitate solution to avoid foaming. h) Incubate samples for 16 to 24 hours in heating unit previously set to 50°C. Remove samples from incubation unit after a minimum of 16 hours. Samples may be prepared for overnight incubation if removed within 24 hours. i) Let samples cool to room temperature. Remove caps and add 12 mL of methanol to each tube using a bottle re-pipettor. j) Dilute each tube to the 50 mL mark with Laboratory Water. k) Cap tubes and mix thoroughly by inversion. l) Attach a 0.45 µ m filter to a 3 or 5 cc syringe and transfer several mL of a sample to the syringe. Filter sample into an autosampler vial, and cap. Repeat with fresh syringe and filter for each sample and blank. m) Samples are ready for analysis or may be frozen for future analysis within 72 hours.
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F. CALCULATIONS
1. Tryptophan Concentration in Samples:
TRP (g/100g sample) = (CTS – CTB) x (volume / SW) x (100g/1000mg/g) Where:
CTS = Concentration of Tryptophan in Sample (mg/L) CTB = Concentration of Tryptophan in Blank (mg/L) Volume = Final volume in Liters i.e. 50 mL = 0.05L SW = Sample Weight in grams
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G. METHOD PERFORMANCE (Data from AMINO-02 AOAC SLV) 1. Accuracy:
Table 1
Accuracy - Spike Recovery
Spike Recovery
Product
SPIFAN Code
Analyte
Nr. replicates (n)
+ Spike Level 2
+ Spike Level 1
Native level
Avg (%) RSD % Avg (%) RSD (%)
SRM1849a
CLC10-B
TRP TRP TRP TRP TRP TRP TRP TRP
12 12 11 12 10 12 12 12
20.1 25.3 18.3 21.4 32.2 19.5 18.3 91.3
99.9
1.8 3.3 8.6 5.2 2.5 3.0 5.1 1.6
100.9 104.1 101.5
2.4 2.7 4.8 3.9 2.7 2.0 3.5 1.3
Infant Formula Powder Partially Hydrolyzed Milk Based Infant Formula Powder Partially Hydrolyzed Soy Based
410057652Z 410457651Z 00859RF00 00795RF K16NTAV E10NWZC 00729RF00
104.3 103.7
Adult Nutritional Powder Low Fat
96.2
99.5
Infant Elemental Powder
100.7 100.5
102.4 101.7
Infant Formula Powder Milk Based Infant Formula Powder Soy Based Adult Nutritional RTF High Fat
93.8
99.5
104.8
104.9
Global Mean % 100.5
3.9 101.8
2.9
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2. Precision:
Table 3
Precision - repeatability.
Product
SPIFAN Code Analyte
Nr. replicates (n) Conc. Level
RSDr
RSD iR
Conc. Level RSDr
RSD iR
REQUIREMENTS SMPR
SRM1849a
CLC10-B Tryptophan
12 12 12 12 12 12 12 12 12 12 12 12 12 12
20.1 25.3 18.3 25.7 24.1 21.4 23.7 32.2 23.2 19.5 18.3 22.4 70.6 91.3
0.4 0.6 5.0 0.8 0.2 0.9 0.9 0.2 2.0 0.3 0.2 0.5 1.9 0.3
1.5
X
≤Z % ≤W%
Infant FormulaPowderPartially Hydrolyzed Milk Based 410057652Z Tryptophan
1.0 Analyte 1 6.9 Analyte 2 2.0 Analyte 3 1.8 Analyte 4 1.7 Analyte 5 1.3 Analyte 1 1.6 Analyte 2 2.4 Analyte 3 1.7 Analyte 4 2.3 Analyte 5 1.8 Analyte 1 3.7 Analyte 2 1.9 Analyte 3 Analyte 4 Analyte 5 Analyte 1 Analyte 2 Analyte 3 Analyte 4 Analyte 5
0.4-5.0
4%
Infant FormulaPowderPartially Hydrolyzed Soy Based
410457651Z Tryptophan 4052755861 Tryptophan 4044755861 Tryptophan 00859RF00 Tryptophan 00866RF00 Tryptophan 00795RF Tryptophan 50350017W1 Tryptophan K16NTAV Tryptophan E10NWZC Tryptophan EV4H2R Tryptophan 00730RF00 Tryptophan 00729RF00 Tryptophan
ToddlerFormulaPowderMilk-Based Infant FormulaPowderMilk-Based Adult Nutritional PowderLow Fat
Child FormulaPowder Infant Elemental Powder
5.0-50
3%
Infant FormulaPowderFOS/GOS Based Infant FormulaPowderMilk Based Infant FormulaPowderSoy Based Infant FormulaRTF Milk Based Adult Nutritional RTF High Prtotein Adult Nutritional RTF High Fat
50-2500
2%
Global Mean %
1.0
2.3
H. SPECIFICITY - Selectivity for the analyte of interest is supported by the following: 1. The selectivity of chromatographic separation combined with native fluorescence 2. Near 100% recovery of reference material
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I. FIGURES FIGURE 1 Typical 5/50 Standard Chromatogram
13.00
12.00
11.00
10.00
TRP_SA - 2.486
9.00
5MDLT - 6.184
8.00
7.00
LU
6.00
5.00
4.00
3.00
2.00
1.00
0.00
8.50
8.00
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
Minutes
FIGURE 2 Typical Blank Chromatogram
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
5MDLT - 6.182
LU
TRP_SA - 2.489
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Minutes
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FIGURE 3 Typical Sample Chromatogram (NIST Standard Reference Material 1849a)
12.00
11.00
10.00
9.00
TRP_SA - 2.483
5MDLT - 6.175
8.00
7.00
6.00
LU
5.00
4.00
3.00
2.00
1.00
3.106
6.875
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.50
6.00
6.50
7.00
7.50
8.00
8.50
5.00
Minutes
2017.03 (JULY 2018) Amino-02 TRP_AOAC_2017.03_MLT_Corrected 080218
FOR ERP USE ONLY DO NOT DISTRIBUTE
J. CONTRIBUTING REFERENCES
1. Narahashi, Y.; Yanagita, M. : (1967) J. Biochemistry 6 pp 633-641. 2. Yamaskov, I.A.; Tichonova, T.V.; Davankov, V.A.; (1986) ENZYME MICROB. TECHNOL. 8 pp241-244.
2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories This method is applicable to the determination of total tryptophan in infant formula and adult/pediatric nutritional formulas. A sample amount targeting about 30 – 50 mg of protein is added to the sample preparation. The limit of quantitation of a typical ready to feed formula is approximately 0.18 mg/100g. A. SAFETY ALL REAGENTS AND EQUIPMENT SHOULD BE HANDLED IN A MANNER CONSISTENT WITH SAFE LABORATORY PRACTICES. REAGENTS AND SAMPLES SHOULD BE TREATED WITH THE RESPECT AND CARE THAT ANY CHEMICAL REQUIRES. REAGENTS WHICH ARE ALLOWED TO COME IN CONTACT WITH THE SKIN, EYES, OR CLOTHING SHOULD BE FLUSHED IMMEDIATELY WITH LARGE VOLUMES OF WATER. IMMEDIATE MEDICAL ATTENTION SHOULD BE OBTAINED IF ANY REAGENT IS INGESTED OR ALLOWED TO COME IN CONTACT WITH THE EYES. ALL OF THE SOLVENTS USED IN THIS METHOD ARE HIGHLY FLAMMABLE AND SHOULD BE KEPT AWAY FROM FLAMES AND OTHER IGNITION SOURCES. METHANOL IS A TOXIC CUMULATIVE POISON THAT CAN CAUSE BLINDNESS. PHOSPHORIC ACID AND HYDROCHLORIC ACID MAY CAUSE IRRITATION TO SKIN AND EYES IF DIRECT CONTACT IS MADE. USE CARE WHEN HANDLING STREPTOMYCES GRISEUS ENZYME: MAY BE HARMFUL IF INHALED. MAY CAUSE RESPIRATORY TRACT, EYE AND SKIN IRRITATION. MAY CAUSE ALLERGIC RESPIRATORY AND SKIN REACTION.
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2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories BA . PRINCIPLE Tryptophan is released (hydrolyzed) from intact protein using a combination of proteolytic enzymes found in pronase, isolated from Streptomyces griseus, for this purpose. The pronase enzyme powder contains at least 10 proteolytic enzymes (depending on the source, supplier etc.), which hydrolyze peptide bonds internally (endoproteases) and externally (exopeptidases), either at the N-terminal end (amino peptidases) or at the carboxy terminus (carboxypeptidases). The protein is thus "attacked" on different sites simultaneously releasing tryptophan in a relatively short period of time. Following proteolysis, free tryptophan is quantitated by reverse phase, isocratic HPLC and fluorescence detection, which provide for a selective and specific determination of tryptophan in nutritional products. The enzymes in pronase self-digest to produce background tryptophan in the absence of sample. Consequently, the enzyme system is non-specific for the sample tryptophan, and a blank subtraction is mandatory. Using this approach, recoveries of free tryptophan spikes as well as tryptophan from BSA spikes are found essentially quantitative, indicating near comparable self-digestion rates with and without sample. Sample preparation consists of adding a weighed sample, the enzyme solution, internal standard (5-methyl-DL-tryptophan) and Trizma buffer into a tube. A small amount of methanol is added as a bactericidal agent. The preparation is mixed and incubated at 50°C for sixteen hours (overnight), to assure complete hydrolysis of all sample types. (While many samples are fully hydrolyzed within 6 hours, some have been found to require longer, thus 16 hours minimum for full applicability). After hydrolysis, the sample-enzyme mixture is diluted to 50 mL with methanol/water and filtered. The sample is injected onto a C-8 column with reference standards and enzyme blank preparations and the analytes of interest detected and quantified fluorometrically. CB . APPARATUS 1. Equipment a) Analytical Column: YMC PAC C8 50 × 3 mm column with 3 micron particle size – Part #OC12S03-053030 – or equivalent. b) HPLC system with autosampler, column oven to maintain 30°C, Fluorescence Detector, binary or quaternary HPLC Pump with degasser, HPLC peak area integrator or chromatography data processor.
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2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories c) Constant temperature oven capable of maintaining 50 (+/- 1) degree Centigrade 2. Instrument Parameters and Conditions a) Mobile Phase Flow Rate : 0.5 mL /minute b) Column Temperature: 30 degrees Centigrade c) Injection volume: 10 µ L. d) Detector settings: Excitation wavelength to 295 nm and emission wavelength to 345 nm e) Typical PMT setting: 7 for Agilent G1321A (see F.3.a) f) Run time: 8.5 minutes, or as needed to fully collect the 5-methyl-DL-tryptophan peak.
E.
D. REAGENTS
1. Standards L-Tryptophan USP Standard Cat #1700501, or equivalent. 2. Chemicals a) Methanol, HPLC grade b) Sodium Phosphate Monobasic, HPLC grade. c) Phosphoric Acid, (85%) d) Trizma Base e) Hydrochloric Acid, 6N.
f) Laboratory Water – minimum of 18 mega ohm conductivity g) 5-Methyl-DL-tryptophan Sigma Cat # M-0534, or equivalent. h) Protease from Streptomyces griseus : Sigma Cat # P-5147, or equivalent. 3. Solutions a) Hydrochloric Acid, 1.0N Solution. b) Trizma Buffer, 0.1M, pH 8.5. c) Protease Solution - Weigh 0.125 ± 0.010 grams to 25 mL with Trizma buffer. Scale volume as needed with proportional weight. Expiration: 6 hours.
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3
2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories d) 5-Methyl-DL-tryptophan, Internal Standard Solution - 0.0400 ± 0.0020 grams to 50 mL with water. Initially add ~25 mL water and stir with small stir bar. Due to slow dissolution, may stir overnight in a refrigerator. Store frozen. Expiration: 6 months e) Tryptophan Stock Standard Solution - (0.0100 ± 0.0010 grams per 100 mL) Store frozen. Expiration: 6 months. Combine the appropriate aliquot of tryptophan and internal standard stock solutions in separate 50 mL volumetric flasks for four working levels. Standard TRP Stock Sol. Int. Std. Stock VERY LOW 0.20 mL 0.5 mL LOW 1.0 mL 0.5 mL MID 5.0 mL 0.5 mL HIGH 10.0 mL 0.5 mL Add 12.0 mL of methanol to each of the four flasks containing the standard/internal standard mixtures, and bring to volume with water. Transfer diluted standards to vials for frozen storage. Expiration: 6 months. g) Mobile Phase: (18:82, Methanol/0.05 M Phosphate Buffer, pH 2.3) h) Column Rinse Solution: (25% methanol in water) PROCEDURE 1. Sample Preparation a) Set heating unit to 50°C prior to experiment. b) Using an analytical balance, tare a 50 mL centrifuge tube. c) Weigh liquid samples and reconstituted powders directly into tarred centrifuge tubes using a disposable transfer pipette. For non-reconstituted powder samples weigh into a tarred tube with a spatula. Record weights. d) Add 0.5 mL (500 µ L) of protease enzyme solution to all samples. F.E. h) Working Standard Solutions:
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2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories e) Prepare no fewer than two "blank" enzyme solutions per run. Blank solutions will contain enzyme, internal standard, and buffer only. Otherwise follow sample preparation. f) Add 0.5 mL of 5-methyl-DL-tryptophan internal standard to all tubes including the enzyme blanks. g) Add 3.0 mL of 0.1M, pH 8.5 Trizma buffer and 200 µ L methanol to all tubes including enzyme blanks. Cap tubes and vortex lightly (about 2 seconds) to mix contents. Be sure powder samples are completely dissolved but do not over agitate solution to avoid foaming. h) Incubate samples for 16 to 24 hours in heating unit previously set to 50°C. Remove samples from incubation unit after a minimum of 16 hours. Samples may be prepared for overnight incubation if removed within 24 hours. i) Let samples cool to room temperature. Remove caps and add 12 mL of methanol to each tube using a bottle re-pipettor. j) Dilute each tube to the 50 mL mark with Laboratory Water. k) Cap tubes and mix thoroughly by inversion. l) Attach a 0.45 µ m filter to a 3 or 5 cc syringe and transfer several mL of a sample to the syringe. Filter sample into an autosampler vial, and cap. Repeat with fresh syringe and filter for each sample and blank. m) Samples are ready for analysis or may be frozen for future analysis within 72 hours.
G.F.
CALCULATIONS
1. Tryptophan Concentration in Samples:
TRP (g/100g sample) = (CTS – CTB) x (volume / SW) x (100g/1000mg/g) Where:
CTS = Concentration of Tryptophan in Sample (mg/L) CTB = Concentration of Tryptophan in Blank (mg/L) Volume = Final volume in Liters i.e. 50 mL = 0.05L SW = Sample Weight in grams
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2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories
GH . METHOD PERFORMANCE (Data from AMINO-02 AOAC SLV) 1. Accuracy:
Table 1
Accuracy - Spike Recovery
Spike Recovery
Product
SPIFAN Code
Analyte
Nr. replicates (n)
+ Spike Level 1
+ Spike Level 2
Native level
Avg (%) RSD % Avg (%) RSD (%)
SRM1849a
CLC10-B
TRP TRP TRP TRP TRP TRP TRP TRP
12 12 11 12 10 12 12 12
20.1 25.3 18.3 21.4 32.2 19.5 18.3 91.3
99.9
1.8 3.3 8.6 5.2 2.5 3.0 5.1 1.6
100.9 104.1 101.5
2.4 2.7 4.8 3.9 2.7 2.0 3.5 1.3
Infant Formula Powder Partially Hydrolyzed Milk Based Infant Formula Powder Partially Hydrolyzed Soy Based
410057652Z 410457651Z 00859RF00 00795RF K16NTAV E10NWZC 00729RF00
104.3 103.7
Adult Nutritional Powder Low Fat
96.2
99.5
Infant Elemental Powder
100.7 100.5
102.4 101.7
Infant Formula Powder Milk Based Infant Formula Powder Soy Based Adult Nutritional RTF High Fat
93.8
99.5
104.8
104.9
Global Mean % 100.5
3.9 101.8
2.9
Page
6
2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories
2. Precision:
Precision - repeatability.
Table 3
SPIFAN Code Analyte
Nr. replicates (n) Conc. Level
RSDr
RSD iR
Conc. Level RSDr
RSD iR
Product
REQUIREMENTS SMPR
SRM1849a
CLC10-B Tryptophan
12 12 12 12 12 12 12 12 12 12 12 12 12 12
20.1 25.3 18.3 25.7 24.1 21.4 23.7 32.2 23.2 19.5 18.3 22.4 70.6 91.3
0.4 0.6 5.0 0.8 0.2 0.9 0.9 0.2 2.0 0.3 0.2 0.5 1.9 0.3
1.5
X
≤Z % ≤W%
Infant FormulaPowderPartially Hydrolyzed Milk Based 410057652Z Tryptophan
1.0 Analyte 1 6.9 Analyte 2 2.0 Analyte 3 1.8 Analyte 4 1.7 Analyte 5 1.3 Analyte 1 1.6 Analyte 2 2.4 Analyte 3 1.7 Analyte 4 2.3 Analyte 5 1.8 Analyte 1 3.7 Analyte 2 1.9 Analyte 3 Analyte 4 Analyte 5 Analyte 1 Analyte 2 Analyte 3 Analyte 4 Analyte 5
0.4-5.0
4%
Infant FormulaPowderPartially Hydrolyzed Soy Based
410457651Z Tryptophan 4052755861 Tryptophan 4044755861 Tryptophan 00859RF00 Tryptophan 00866RF00 Tryptophan 00795RF Tryptophan 50350017W1 Tryptophan K16NTAV Tryptophan E10NWZC Tryptophan EV4H2R Tryptophan 00730RF00 Tryptophan 00729RF00 Tryptophan
ToddlerFormulaPowderMilk-Based Infant FormulaPowderMilk-Based Adult Nutritional PowderLow Fat
Child FormulaPowder Infant Elemental Powder
5.0-50
3%
Infant FormulaPowderFOS/GOS Based Infant FormulaPowderMilk Based Infant FormulaPowderSoy Based Infant FormulaRTF Milk Based Adult Nutritional RTF High Prtotein Adult Nutritional RTF High Fat
50-2500
2%
Global Mean %
1.0
2.3
Page
7
2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories
HJ . FIGURES FIGURE 1 Typical 5/50 Standard Chromatogram
13.00
12.00
11.00
10.00
TRP_SA - 2.486
9.00
5MDLT - 6.184
8.00
7.00
LU
6.00
5.00
4.00
3.00
2.00
1.00
0.00
8.50
8.00
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
Minutes
FIGURE 2 Typical Blank Chromatogram
Page
8
2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories
0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50
5MDLT - 6.182
LU
TRP_SA - 2.489
8.50
8.00
7.50
7.00
6.50
6.00
5.50
5.00
4.50
4.00
3.50
3.00
2.50
2.00
1.50
1.00
0.50
Minutes
FIGURE 3 Typical Sample Chromatogram (NIST Standard Reference Material 1849a)
12.00
11.00
10.00
9.00
TRP_SA - 2.483
5MDLT - 6.175
8.00
7.00
6.00
LU
5.00
4.00
3.00
2.00
1.00
3.106
6.875
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
4.50
5.00
5.50
6.00
6.50
7.00
7.50
8.00
8.50
Minutes
Page
9
2017.03 (JULY 2018) Amino-02 Tryptophan by enz hyd_Abbott Labs_Method
FOR ERP USE ONLY DO NOT DISTRIBUTE HPLC Determination of Total Tryptophan in Infant Formula and Adult/Pediatric Nutritional Formula Following Enzymatic Hydrolysis Abbott Nutrition, Division of Abbott Laboratories 1. Narahashi, Y.; Yanagita, M. : (1967) J. Biochemistry 6 pp 633-641. 2. Yamaskov, I.A.; Tichonova, T.V.; Davankov, V.A.; (1986) ENZYME MICROB. TECHNOL. 8 pp241-244. JL . SPECIFICITY - Selectivity for the analyte of interest is supported by the following: 1. The selectivity of chromatographic separation combined with native fluorescence 2. Near 100% recovery of reference material IK . CONTRIBUTING REFERENCES
Page
10
2017.03 (JULY 2018) Amino-02 AOAC_TRP_MLT_- AOAC_BlindDup_00729RF00
FOR ERP USE ONLY DO NOT DISTRIBUTE
8/5/2018 Copyright 2006,2012 by AOAC International, all rights reserved.
AOAC International Interlaboratory Study Workbook
Version:
2.1
Blind (Unpaired) Replicates INSTRUCTIONS
1. SAVE WORKBOOK UNDER STUDY NAME & SAMPLE NUMBER 1.1. After opening the workbook, use File|Save As to save under a new name indicative of the study and sample number.
2. ENTER DATA:
2.1. Select the Data worksheet. 2.2. Enter information in YELLOW, boxed cells ONLY! 2.3. Leave unused cells empty. 2.4. Do NOT delete or insert Rows or Columns!
2.5. To remove data, select the range of cells and press the "Delete" key. 2.6. To paste data from another source, use Edit|Paste Special|Values. 2.7. Save your workbook to protect against loss.
3. REVIEW RESULTS:
3.1. Select the Results worksheet. 3.2. Evaluate data by examining statistics on the Results worksheet. 3.3. If laboratory outliers are found, remove the related information on the Data sheet (see Step 1.5 above). 3.4. Save your workbook to protect against loss.
4. REPORT RESULTS
4.1. Select the Report worksheet. 4.2. Print and report the Report worksheet.
5. SAVE DATA:
5.1. Once you are confident the analysis is complete, save the workbook.
2017.03 (JULY 2018) Amino-02 AOAC_TRP_MLT_- AOAC_BlindDup_00729RF00
FOR ERP USE ONLY DO NOT DISTRIBUTE
AOAC International Interlaboratory Study Workbook
Version:
2.1
Blind (Unpaired) Replicates Data Sheet Name of study: Date of study:
AOAC 2017.03 Determination of L-Tryptophan
8/24/2017
Sample ID:
00729RF00 - DYLB360/ZMQM883
Factor for Units of measurement:
1.00E-05 (% = 1E-2, mg/100g = 1E-5, ppm = 1E-6, ppb = 1E-9, etc.)
INSTRUCTIONS:
1. Enter information in YELLOW, boxed cells ONLY! 2. Leave unused cells empty. 3. Do NOT delete or insert Rows or Columns! 4. To remove data, select the range of cells and press the "Delete" key. 5. The worksheet is protected: To paste values from another sheet, use Edit|Pas
Replicate Replicate Replicate Replicate Replicate
Laboratory
1
2
3
4
5
1 85.565 86.314 2 85.604 89.293 3 89.611 90.620 4 91.000 93.300 5 89.167 86.762 6 94.467 95.841 7 86.028 85.304 8 94.276 94.687 9 91.622 91.913 10 94.055 94.994
11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
2017.03 (JULY 2018) Amino-02 AOAC_TRP_MLT_- AOAC_BlindDup_00729RF00
FOR ERP USE ONLY DO NOT DISTRIBUTE
AOAC International Interlaboratory Study Workbook
Version:
2.1
Blind (Unpaired) Replicates Results Sheet Seq.
Item
Symbol
Value
Study name: Study date: Sample ID:
AOAC 2017.03 Determination of L-Tryptophan
8/24/2017
00729RF00 - DYLB360/ZMQM883
1 Total number of laboratories 2 Total number of replicates 4 Sum of replicates squared 5 Sum of lab totals 6 Sum of lab averages 7 Sum of lab totals squared/n(L) 8 Sum of squares for replicates 9 Sum of Squares for Labs 3 Average number of replicates per lab
p
10 20
Sum(n(L)) Sum(n(L))/p Sum(n(L)^2)
2.00
40
Sum(Lab Totals) Sum(Lab Avg.) Sum(T(L)^2/n(L))
1810.42 905.21 14.91 236.17 2.0000 90.5211 90.5211 1.4907 1.2209 12.3753 13.8659 3.7237 1.35 4.11 3.419 10.426
164117.60
SSQ(devn.)
LSS
10 Effective mean number of reps per lab 11 Unweighted mean of the laboratory averages 12 Overall mean of all data (grand mean) 14 Repeatability standard deviation 15 "Pure" among laboratories variance 17 Reproducibility standard deviation 18 Repeatability relative standard deviation 19 Reproducibility relative standard deviation 16 Reproducibility variance 13 Repeatability variance
NBAR XBAR
XBARBAR
s(r)^2
s(r)
s(L)^2 s(R)^2
s(R)
RSD(r) RSD(R)
20 Repeatability value = 2.8*s(r) 21 Reproducibility value = 2.8*s(R)
r
R
0.72
22 HORRAT value
Record these values for Results Number of Laboratories Largest within variance Largest average lab result Smallest average lab result
10
6.8057 95.1542 85.6663
If proportion of labs removed from the total is less than 2/9, then Cochran's Test =
45.7% 65.5%
Critical value, 2.5% significance =
Result =
Not significant
with maximum within lab variance for
Lab 2
If Cochran test is not significant, then proceed
6.4% 42.0%
Single Grubb's Test =
Critical value, 2.5% significance =
Result =
Not significant
highest average at
Lab 6
gives % decrease in standard deviation =
5.2%
lowest average at
Lab 7
gives % decrease in standard deviation =
6.4%
If Single Grubb's test is not significant, then proceed Double Grubb's Test =
17.6% 56.1%
Critical value, 2.5% significance =
Result =
Not significant
Excluding the highest avg. and next highest avg gives % decrease in standard deviation = Excluding the lowest avg. and next lowest avg gives % decrease in standard deviation = Excluding the highest avg. and lowest avg gives % decrease in standard deviation =
11.9%
17.6%
10.8%
2017.03 (JULY 2018) Amino-02 AOAC_TRP_MLT_- AOAC_BlindDup_00729RF00
FOR ERP USE ONLY DO NOT DISTRIBUTE
AOAC International Interlaboratory Study Workbook
Version:
2.1
Blind (Unpaired) Replicates Study Reported Values
Seq.
Item
Symbol
Value
Study name: Study date: Sample ID:
AOAC 2017.03 Determination of L-Tryptophan
24-Aug-2017
00729RF00 - DYLB360/ZMQM883
1 Total number of laboratories 2 Total number of replicates
p
10 20
Sum(n(L)) XBARBAR
3 Overall mean of all data (grand mean) 4 Repeatability standard deviation 5 Reproducibility standard deviation 6 Repeatability relative standard deviation 7 Reproducibility relative standard deviation
90.5211 1.2209 3.7237
s(r) s(R)
RSD(r) RSD(R)
1.35 4.11
8 HORRAT value
0.72
2017.03 (JULY 2018) Amino-02 AOAC_TRP_MLT_- AOAC_BlindDup_00729RF00
FOR ERP USE ONLY DO NOT DISTRIBUTE
AOAC International Interlaboratory Study Workbook
Version:
2.1
Blind (Unpaired) Replicates Calculations Sheet
AOAC 2017.03 Determination of L-Tryptophan
Summary Statistics
6.8057 Largest within variance 95.1542 Largest average lab result 85.6663 Smallest average lab result 90.5211 Grand average of all lab averages
3.19087 S(2H)
95.1542 Max Val(1) 94.5242 Max Val(2) 85.6663 Min Val(1) 85.9397 Min Val(2) 3.2303 S(HL) 95.1542 Max Val 85.6663 Min Val 2.9856 S(2L)
3.62224 s
3.43210 S(h) 3.38927 S(l)
95.154229 85.666292
10 # Laboratories
Average Lab Result^2
Average Lab Result^2 Results^2
Average Lab Result
Sum of Lab Results
# Lab Replicates given
Sum of Results^2/#Reps DEVN of Sq
Variance within DEVN of Sq
Lab
1 2 3 4 5 6 7 8 9
2 171.8794 85.9397 7385.6308 14771.5418 14771.2617 2 174.8966 87.4483 7647.2065 15301.2188 15294.4130 2 180.2309 90.1155 8120.7961 16242.1009 16241.5921 2 184.3000 92.1500 8491.6225 16985.8900 16983.2450 2 175.9283 87.9642 7737.6918 15478.2762 15475.3836 2 190.3085 95.1542 9054.3273 18109.5988 18108.6547 2 171.3326 85.6663 7338.7135 14677.6893 14677.4270 2 188.9632 94.4816 8926.7722 17853.6291 17853.5444 2 183.5343 91.7672 8421.2136 16842.4695 16842.4272 2 189.0484 94.5242 8934.8254 17870.0918 17869.6509
0.2802 0.2802 6.8057 6.8057 0.5088 0.5088 2.6450 2.6450 2.8926 2.8926 0.9441 0.9441 0.2623 0.2623 0.0847 0.0847 0.0423 0.0423 0.4410 0.4410
0.2802 Lab 1 6.8057 Lab 2 0.5088 Lab 3 2.6450 Lab 4 2.8926 Lab 5 0.9441 Lab 6 0.2623 Lab 7 0.0847 Lab 8 0.0423 Lab 9
85.9397 Lab 1 87.4483 Lab 2 90.1155 Lab 3 92.1500 Lab 4 87.9642 Lab 5 95.1542 Lab 6 85.6663 Lab 7 94.4816 Lab 8 91.7672 Lab 9
10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49
0.4410 Lab 10 94.5242 Lab 10
Lab 11 Lab 12 Lab 13 Lab 14 Lab 15 Lab 16 Lab 17 Lab 18 Lab 19 Lab 20 Lab 21 Lab 22 Lab 23 Lab 24 Lab 25 Lab 26 Lab 27 Lab 28 Lab 29 Lab 30 Lab 31 Lab 32 Lab 33 Lab 34 Lab 35 Lab 36 Lab 37 Lab 38 Lab 39 Lab 40 Lab 41 Lab 42 Lab 43 Lab 44 Lab 45 Lab 46 Lab 47 Lab 48 Lab 49
Lab 11 Lab 12 Lab 13 Lab 14 Lab 15 Lab 16 Lab 17 Lab 18 Lab 19 Lab 20 Lab 21 Lab 22 Lab 23 Lab 24 Lab 25 Lab 26 Lab 27 Lab 28 Lab 29 Lab 30 Lab 31 Lab 32 Lab 33 Lab 34 Lab 35 Lab 36 Lab 37 Lab 38 Lab 39 Lab 40 Lab 41 Lab 42 Lab 43 Lab 44 Lab 45 Lab 46 Lab 47 Lab 48 Lab 49
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