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ChromaDex Analytics Analytical Test Report

Turkesterone in Mass FX for Thermolife International
Report Number: CDXA-ATR-957-00 Project Number: ORD24060 Part Number: CDA-00100289-ATR Revision: 00 Issue Date: 21 Aug 2008 Page 1 of 10

Prepared By:
James Traub
LC-MS Chemist

Date

Reviewed By:
Seth Nosel
Quality Control

Date
Digitally signed by Steve Baugh DN: CN = Steve Baugh, C = US, O = ChromaDex Analytics, OU = CDXA Reason: I am approving this document Date: 2008.08.21 17:38:25 -06'00'

Approved By:

Steve Baugh
Steve Baugh

Date

Manager, Analytical Services

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 be applied to any other material or sample.

Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 2 of 10 1. OBJECTIVE To screen for the presence of Turkesterone in Mass FX sample.

2.

SAMPLE(S) 2.1. Mass FX (Lot 47728 Feb2011, CDXA-08-1372)

3.

STANDARD(S) 3.1. Turkesterone standard (CDXA-08-1130)

4.

SOLVENTS AND REAGENTS 4.1 4.2 4.3 Deionized water, 18 M Milli-Q Methanol, EMD, Lot 48109 Acetonitrile, EMD, Lot 48158

5.

EQUIPMENT 5.1 Agilent 1100 Series HPLC System with Diode Array Detector (DAD) 5.2 Agilent G2445D SL Ion Trap Mass Spectrometer with Electrospray Ionization (ESI) Source 5.3 Agilent Zorbax Eclipse XDB-C18 Column (150 x 4.6 mm) 5.4 Mettler Toledo Analytical Balance (CDXA #000034) 5.5 Assorted Volumetric and Laboratory Glassware 5.6 VWR Ultrasonic Bath (CDXA #000408) 5.7 Syringes and filters (PTFE, 0.45 m) 5.8 HPLC Vials and Caps

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 3 of 10 6. STRUCTURES

OH

HO

OH

OH

HO H HO H O Turkesterone C27H44O8 Exact Mass: 496.30

7. SAMPLE PREPARATION 7.1. Approximately one gram of powdered sample was weighed into a 10 mL volumetric flask, 8 mL of methanol added, and the sample sonicated for 30 minutes. After sonication the sample was allowed to return to room temperature, diluted to volume with methanol, and then filtered through a 0.45 m PTFE filter into an HPLC vial for analysis.

OH

8.

STANDARD PREPARATION 8.1. Approximately 1 mg of Turkesterone standard was weighed into a 1 mL volumetric flask and diluted to volume with methanol. The sample was mixed on a vortex mixer for 30 seconds, sonicated for 5 minutes, and the solution allowed to return to room temperature . The standard solution was then used to prepare linearity standards.

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 4 of 10 9. CALIBRATION 9.1. A three point calibration curve was generated using peak areas at 205 nm. Linear fit was = 0.99991. Actual concentrations of standards are listed below. Standard ID Standard 1 Standard 2 Standard 3
Area

Concn. (mg/mL) 0.008 0.080 0.800

Turkesterone, DAD1 A Area = 1806.55984*Amt -6.7558577 Rel. Res%(1): 70.976

1400 1200

1000

800 600 400

200 1 0 0

2 Correlation: 0.99991 0.2 0.4 0.6 Amount[mg/ml]

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 5 of 10 10. ANALYTICAL METHOD 10.1. HPLC HPLC Mobile Phases Mobile Phase Gradient Agilent 1100 HPLC with DAD Detection A = Water B = Acetonitrile time 0 15 20 25 27 35 %B 5 35 95 35 5 5

Column Flow Rate Injection Volume Temperature UV Detection 10.2. Mass Spectrometer Mass Spectrometer Ion Source Type Ion Polarity Nebulizer Pressure Dry Gas Temperature Average Target ions

Zorbax Eclipse XDB-C18; 150 x 4.6 mm 1.000 mL/min 10 L 25 C 205 + 4 nm

Agilent G2445D SL Ion Trap Mass Spectrometer with ESI Source ESI (Electrospray) Positive 45 psi 8 L/min 350oC 5 spectra [M + H]+ = 497.2 m/z

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 6 of 10 11. RESULTS 11.1. The described method (CDXA-ATM-029-01-01) is capable of quantitating Turkesterone in the range 80.0 ppm 8,000 ppm, as determined below: For a 1.0 gram sample prepared at a final volume of 10 mL, and a low standard of 8.0 g/mL and a high standard of 800 g/mL, we have determined the range of quantitation for Turkesterone in a sample as follows: 8.0 g/mL 1.0 g / 10 mL = 80.0 ppm

800 g/mL = 8,000 ppm 1.0 g / 10 mL 11.2. By UV detection at 205 nm, no Turkesterone was detected at 80 ppm concentration in Mass FX (CDXA-08-1372). Mass spectral analysis indicates that Turkesterone may be present at very low concentration (ions 497.2, 479.2, 461.2, 443.2 m/z), but this result could not be confirmed by UV signature of the compound, nor quantitated by UV detection.

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 7 of 10 12. CHROMATOGRAMS Figure 1 Turkesterone Standard (CDXA-08-1130): HPLC Chromatogram (205 nm) Mass Spectrum at 8.9 min
DAD1 A, Sig=205,4 Ref=600,100 (G1408\G1408_04.D) mAU 120 8.887 - Turkesterone 0 5

100

80

60

40

20

0 10 15 20 25 30 min

Intens. x10 6 2.0

+M S, 8.9m i n (#890)

479.2

1.5

1.0 542.2

461.2 0.5

443.2

497.2 956.9

0.0 100 200 300 400 500 600 700 800 900 m /z

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 8 of 10 Figure 2 Mass FX (Lot 47728 Feb2011, CDXA-08-1372): HPLC Chromatogram (205 nm) Mass Spectrum at 8.9 min
DAD1 A, Sig=205,4 Ref=600,100 (G0908\G0908_07.D) mAU 700

600

500

400

300

200

100

0 0 5 10 15 20 25 30 min

Intens. x10 6 5 479.2

+M S, 9.2m i n (#952)

461.1

2 497.2 443.1 1 595.0 227.0 176.9 0 100 200 300 400 500 600 700 800 900 m /z 311.0 341.1 387.1 535.1 685.1

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 9 of 10 Figure 3 UV Spectrum of Turkesterone Standard (CDXA-08-1130):
DAD1, 8.887 (333 mAU,Apx) of G1408_04.D mAU 300

250

200

150

100

50

0 200 225 250 275 300 325 350 375 nm

Figure 4 UV Spectrum of Mass FX Sample (Lot 47728 Feb2011, CDXA-081372):

DAD1, 8.966 (48.6 mAU, - ) of G0908_07.D mAU

40

30

20

10

0 200 225 250 275 300 325 350 375 nm

13.

CALCULATIONS 13.1. Standard Purity Compensation Ms x Ps Where: Ms = Mass of Standard in Mixed Standard Ps = Purity of Standard in Mixed Standard

13.2. Linear Regression Plot the average peak area vs. concentration (mg/mL) for the Standard Solution chromatograms. Linear regression must be used to calculate the slope, intercept, and correlation coefficient for each compound. The regression line will have an equation of the type:

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Turkesterone in Mass FX for Thermolife International CDXA-ATR-957-00 Page 10 of 10 y = mx + b Where: y m x b = = = = Peak area of the component Slope of the regression line Concentration of the component y-Intercept of the regression line

Note: Most chromatography data software will automatically perform linear regression to determine the calibration curve, and calculate the slope, intercept, and correlation coefficient.

14.

REFERENCES ChromaDex Analytical Method (CDXA-ATM-029-01-01) ChromaDex Analytical Lab Notebook (CDXA111 p.63) ChromaDex Analytical Lab Notebook (CDXA118 p.47)

15.

HISTORY OF REVISIONS Date 21 Aug 2008 Revision 00 Title/Changes New: CDXA-ATR-957-00 Turkesterone in Mass FX for Thermolife International

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