Prepared By:
Senior Analytical Chemist
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Quality Assurance
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Analytical Test Report
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Test Location:
Boulder, CO.
Assay:
Amino Acids by HPLC
SUMMARY
SAMPLE(S)
Lot # CDXA #
RESULTS
Table 1 –CDXA-11-0407
Analyte Units Spec. Result Reporting
Limit
L-Leucine mg per tablet 613.6 – 920.40 808
L-Isoleucine mg per tablet 96.00 – 144.00 121
L-Valine mg per tablet 96.00 – 144.00 116
Tablet Weight mg per tablet 1550
ND – Not detected
ANALYTICAL METHOD
STANDARD(S)
All standards supplied by ChromaDex, unless otherwise specified.
LABORATORY SUPPLIES
Analytical Balance Ultrasonication Bath
Assorted and Volumetric glassware Syringes and Syringe Filters HPLC glass vials and caps
SOLVENTS AND REAGENTS
Milli-Q Water Methanol (MeOH)
Sodium Phosphate, Monobasic (NaH2PO4)
Sodium Hydroxide Solution (NaOH) Acetonitrile (ACN)
Hydrochloric acid (HCl)
OPA (o-phthalaldehyde) – Derivatization reagent for primary amino acids
FMOC (9-fluorenyl-methyl chloroformate) – Derivatization reagent for secondary amino acids Borate buffer (0.4N in water)
SOLUTION PREPARATION
Diluent – 0.1 N HCl
The diluent was prepared by transferring 8 mL of HCl to a 1000 mL volumetric flask and diluting it to volume with H2O.
Stock Solution - 400mM NaH2PO4
Solution was prepared by adding 55 grams NaH2PO4 to 900 mL of water and stirring
until completely dissolved. The solution was transferred to a 1,000 mL volumetric flask, the flask brought to mark with water, and the solution filtered (0.45 µm Supor).
Part #
STANDARD PREPARATION
Amino Acid Mix Stock Standard – Includes Leucine, Isoleucine, and Valine
A mixed amino acid standard containing 17 amino acids was purchased from Agilent and arrived in 0.1N HCl. Calibration standards were then prepared from this mixed stock.
SAMPLE PREPARATION
Weighed about 50 mg of sample into a 50 mL volumetric flask. Diluted to volume with 0.1 N HCl and sonicated for 30 minutes. The solution was further diluted 1:10 with diluent. An aliquot of the original sample solution and the diluted sample solution were filtered using a 0.45 µm PTFE syringe filter and the filtrate collected in an HPLC vial for analysis
INSTRUMENT PARAMETERS
Instrument Agilent 1100 Series HPLC System
Detection UV-Vis
Mobile Phase A 40 mM NaH2PO4, pH 7.8
Mobile Phase B 45:45:10 ACN-MeOH-Water
Gradient Program Time (min) %A %B
0.0 100 0 1.0 100 0 9.80 43 57 10.0 0 100 12.0 0 100 12.5 100 0 14.0 100 0
Column Agilent Zorbax Eclipse-AAA; 75 x 4.6 mm, 3.5 µm
Flow Rate 2.0 mL/min
UV Detection 338 nm
Injection Volume Injector Program
DATA
FIGURES
Figure 1: Amino acid Mix Standard (UV Chromatogram)
min 0 1 2 3 4 5 6 7 8 9 mAU 0 20 40 60 80 VWD1 A, Wavelength=338 nm, TT (A2511A\A2511_04.D) 6.57 0 - V aline 7.46 8 - I soleu cine 7.81 2 - L eucin e 10.2 20 10.6 77 10.8 51 11.2 16 11.4 18 11.4 95 11.790 12.409 12.776 13.2 34 1 3.44 5
Figure 2: Sample CDXA 1234 – High Concentration (UV Chromatogram)
min 0 1 2 3 4 5 6 7 8 9 mAU 0 5 10 15 20 25 VWD1 A, Wavelength=338 nm, TT (A2511A\A2511_09.D) Area: 58.4537 Area: 54 .8491 6.57 7 - V aline 7.47 6 - I soleu cine
Figure 3: Sample CDXA 1234– Low Concentration (UV Chromatogram)
mAU 20 25 VWD1 A, Wavelength=338 nm, TT (A2511A\A2511_10.D) 10.6 81 10.8 56 11.2 22 11.4 30
REFERENCES
Analytical Method: 99.1-CD-2.0-000186 “Amino Acids by Pre-Column Derivitization HPLC.”
Laboratory Notebook Page(s)
239 66
185 62
242 162 - 163
REVISION HISTORY
Revision Number Document/Changes
