Analytical Test Report Customer: Address (City, State): Purchase Order: Report Number: Project Number: Date Received: Date of Report: Test Location: Boulder, CO. Assay: Part Number: Amino Acids by HPLC CDA-00100666-ATR Prepared By: Senior Analytical Chemist Date Reviewed By: Quality Assurance Date Approved By: Manager, Analytical Services Date Signed original on file at CDXA This product analysis is subject to our Standard Terms and Conditions for the Purchase and Sale of ChromaDex Products and or Services, a copy of which has been provided to our client and is incorporated herein by this reference. As more specifically set forth therein, this product analysis is for the benefit of our client only, may not be relied upon by any other party without our prior written consent, relates solely to the sample(s) provided to us by our client and therefore cannot by applied to any other material or sample.
CDXA-ATR-2936-00 Page 2 of 6 SUMMARY SAMPLE(S) Lot # CDXA # RESULTS Table 1 CDXA-11-0407 Analyte Units Spec. Result 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 Reporting Limit Tablet Weight mg per tablet 1550 ND Not detected BRL Below reporting limit (compound detected below RL)
CDXA-ATR-2936-00 Page 3 of 6 ANALYTICAL METHOD STANDARD(S) All standards supplied by ChromaDex, unless otherwise specified. Part # Amino Acid Mix Snap-n-shoot Agilent # BCBC7701 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 (NaH 2 PO 4 ) 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 H 2 O. Stock Solution - 400mM NaH 2 PO 4 Solution was prepared by adding 55 grams NaH 2 PO 4 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). Mobile Phase A - 40mM NaH 2 PO 4, ph 7.8 Solution was prepared by adding 200 ml of 400 mm NaH 2 PO 4 to a 2,000 ml volumetric flask, adding 1,500 ml of water, and setting the ph to 7.8 with 1.0 N NaOH. The flask was then brought to mark with water. Mobile Phase B - 45:45:10 ACN-MeOH-Water Solution was prepared by adding 900 ml Acetonitrile and 900 ml Methanol to a 2,000 ml volumetric flask and then bringing the flask to mark with water.
CDXA-ATR-2936-00 Page 4 of 6 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 Detection Agilent 1100 Series HPLC System UV-Vis Mobile Phase A 40 mm NaH 2 PO 4, 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 Temperature 40 o C
13.241 12.797 12.429 11.799 13.443 7.825- Leucine 11.506 11.430 10.681 10.856 11.222 6.577- Valine 7.476- Isoleucine 6.570- Valine 7.468- Isoleucine 7.812- Leucine 10.220 13.234 11.790 12.776 12.409 13.445 11.495 11.418 11.216 10.677 10.851 CDXA-ATR-2936-00 Page 5 of 6 DATA FIGURES Figure 1: Amino acid Mix Standard (UV Chromatogram) mau VWD1 A, Wavelength=338 nm, TT (A2511A\A2511_04.D) 80 60 40 20 0 0 1 2 3 4 5 6 7 8 9 min Figure 2: Sample CDXA 1234 High Concentration (UV Chromatogram) mau VWD1 A, Wavelength=338 nm, TT (A2511A\A2511_09.D) 25 20 15 Area: 58.4537 Area: 54.8491 10 5 0 0 1 2 3 4 5 6 7 8 9 min Figure 3: Sample CDXA 1234 Low Concentration (UV Chromatogram) mau VWD1 A, Wavelength=338 nm, TT (A2511A\A2511_10.D) 25 20 15 10 5 0 0 1 2 3 4 5 6 7 8 9 min
CDXA-ATR-2936-00 Page 6 of 6 REFERENCES Analytical Method: 99.1-CD-2.0-000186 Amino Acids by Pre-Column Derivitization HPLC. Laboratory Notebook 239 66 185 62 Page(s) 242 162-163 REVISION HISTORY Revision Number Document/Changes 00 New report