Testing Report for

JON KRAKAUER

CONFIDENTIAL REPORT

Jon Krakauer
Prepared By: Avomeen Analytical Services
4840 Venture Drive Ann Arbor, MI 48108 Date: September 6, 2013

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ODAP Analysis in Hedysarum Alpinum

To:

Jon Krakauer

Analysis for ODAP
Thank you for contacting Avomeen Analytical Services for the testing for beta-N-oxalyl-L-alpha-betadiaminopropionic acid (ODAP) in Hedysarum alpinum Seed samples. Following are the results, methodology, and data associated with the testing for and quantification of ODAP.

Avomeen ID 082913JO1777 082913JO1778

Table 1. Sample Information Sample Description Hedysarum Alpinum Seeds ODAP

Lot # 8/24/13 N/A

Figure 1. “As Received” Samples

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ODAP Analysis in Hedysarum Alpinum

Executive Summary
We have determined the quantity of ODAP extracted from the Hedysarum alpinum seeds using 0.2 M perchloric acid solution to be 3.94 mg / g of pulverized dried seed.

Analytical Methods
Sample Preparation: Hedysarum alpinum seeds were crushed to a powder in an SPEX 6770 Freezer Mill. Extraction was performed on the powdered seed (0.1623 g) using 5 mL of 0.2 M perchloric acid solution (aqueous) at 0 °C for 1 hr. The sample was then centrifuged for 15 min. at 0 °C to separate the suspended insoluble. The extracts were derivatized with a para-nitrobenzoyl tag to enhance the signal in the UV-Visible range. Tagging was achieved using 1200 µL of 0.025 M para-nitrobenzoyl chloride (PNZ-Cl) in acetonitrile, 300 µL of 0.5 M sodium bicarbonate (aqueous), and 500 µL of the centrifuged extract solution. The solution was vortexed for 1 min. followed by sonication at room temperature for 5 min. The resulting mixture was then filtered through a 0.45 µm nylon filter prior to injection into the HPLC. Preparation of Standard Solutions: A 2.00 mM ODAP solution was prepared by weighing 8.8 mg of ODAP and diluting to 25 mL with deionized water in a volumetric flask. A 400 µM solution was prepared by dilution of a 2.0 mL aliquot of the 2.00 mM ODAP solution to 10 mL. A 200 µM solution was prepared by dilution of a 1.0 mL aliquot of the 2.00 mM ODAP solution to 10 mL. A 40 µM solution was prepared by dilution of a 1.0 mL aliquot of the 400 µM ODAP solution to 10 mL. The ODAP was then derivatized using 1200 µL of 0.025 M para-nitrobenzoyl chloride (in acetonitrile), 300 µL of 0.5 M sodium bicarbonate (aqueous), and 500 µL of the corresponding ODAP solution. The solutions were vortexed for 1 min. followed by sonication at room temperature for 5 min. to give standard solutions with concentrations of 500 µM, 100 µM, 50 µM, and 10 µM of the analyte. The resulting solutions were then filtered through a 0.45 µm nylon filter prior to injection into the HPLC. HPLC Analysis for Quantification of ODAP extracted from JO1777: An Agilent 1100 HPLC was used for the analysis of the ODAP standard solutions and extract. The procedure used is a slight modification of the one used by Li, et. al. (Z.Y. Yan, C.J. Jiao, Y.P. Wang, F.M. Li, Y.M. Liang, Z.X. Li, Analytica Chimica Acta 543 (2005) 199-205). The system parameters are found below:  Column: Supelco Discovery® C18 (4.6 x 250 mm, 5µm) equipped with a C18 guard column (4.6 x 10 mm)  Injection Volume: 10 μL  Column Temperature: 40°C  Flow Rate: 1.0 mL/min  Wavelength: 260 nm Table 2. HPLC Mobile Phase Gradient % 0.1 M Sodium Acetate % Acetonitrile 75% 25% Ramp to 65% Ramp to 35% Ramp to 37.5% Ramp to 62.5% Ramp to 0% Ramp to 100%

Time 0 0-2 2-20 20-26

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ODAP Analysis in Hedysarum Alpinum

Results and Discussion
Using the peak areas measured for the peak at 2.7 min. with sample concentrations of approximately 500 µM, 100 µM, 50 µM, and 10 µM (Table 3), a linear calibration curve relating concentration to peak intensity was developed (Figure 2). Chromatographs of the acetonitrile blank, PNZ-Cl standard solution, and the standard samples are shown in Figures 3 – 8.

Table 3. ODAP Stardard Concentrations and Relative Peak Areas Measured through HPLC Analysis Concentration (uM) 499.631 99.9262 49.9631 9.99262 Peak Area 2866.7 595.3 309.9 73.3

3500 3000 2500 Peak Area y = 5.69483x + 22.34587 R² = 0.99999

2000
1500 1000 500 0 0 200 400 600 Concentration (uM) Series1 Linear (Series1)

Figure 2. Calibration Curve using ODAP Standards Using the calibration curve, the concentration of ODAP in the extracted sample was calculated using the peak area from the chromatogram (Figure 9). The resulting concentration was then used to calculate the amount of material extracted from the powdered seed. These results indicate 3.94 mg of ODAP extracted / g of powdered Hedysarum alpinum seed. In order to confirm the peak identification a UV-Visible scan (200 – 400 nm) of the peak at 2.7 min. was conducted on both the 500 µM standard and the extracted sample. Two peaks were identified with λ-Max of approximately 220 nm and 270 nm. The UV/Vis data is shown in Figures 10 and 11.

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ODAP Analysis in Hedysarum Alpinum

Figures

Figure 3. HPLC Chromatograph of Acetonitrile Blank

Peaks from PNZ-Cl

Figure 4. HPLC Chromatograph of para-Nitrobenzoyl Chloride (PNZ-Cl)

Peaks from PNZ-Cl ODAP

Figure 5. HPLC Chromatograph of 500 µM ODAP Standard

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ODAP Analysis in Hedysarum Alpinum

Peaks from PNZ-Cl

ODAP

Figure 6. HPLC Chromatograph of 100 µM ODAP Standard

Peaks from PNZ-Cl

ODAP

Figure 7. HPLC Chromatograph of 50 µM ODAP Standard

Peaks from PNZ-Cl

ODAP

Figure 8. HPLC Chromatograph of 10 µM ODAP Standard

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ODAP Analysis in Hedysarum Alpinum

Peaks from PNZ-Cl

ODAP

Figure 9. HPLC Chromatograph of Hedysarum alpinum Extract

λ-Max Approx. 270 nm λ-Max Approx. 220 nm

Figure 10. UV/Visible Spectrum of Peak at 2.7 min. in Chromatograph of 500 µM ODAP Standard

λ-Max Approx. 270 nm λ-Max Approx. 220 nm

Figure 11. UV/Visible Spectrum of Peak at 2.7 min. in Chromatograph of Hedysarum alpinum Extract

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ODAP Analysis in Hedysarum Alpinum

References
Z.Y. Yan, C.J. Jiao, Y.P. Wang, F.M. Li, Y.M. Liang, Z.X. Li, Analytica Chimica Acta 543 (2005) 199-205

Description of Instrumentation Used
Reverse Phase High Performance Liquid Chromatography (rpHPLC): Avomeen uses Agilent 1100 series instruments with quaternary pumps. Detection modules include Refractive Index, UV variable wavelength and UV Diode Array Detectors. The quaternary pumps allow the use of variable solvent chemistry to optimize resolution and run time. Diode Array detection allows for collection of data from multiple wavelengths simultaneously. The system is optimized for reverse phase chromatography, which allows the analyst to utilize a highly polar mobile phase solution that carries the compound of interest though the chromatography column. The column contains a non-polar stationary phase that interacts with the compound of interest as it is pumped through the column. The compound eventually is released from the column and travels to the detector where a signal arises based on the compound’s characteristic absorbance and retention time. The analog signal is converted to a usable chromatogram where the information about the compound can be analyzed with high accuracy and precision.

Wrap Up
Thank you for consulting with Avomeen Analytical Services. If you have any questions regarding this analysis, or if we can be of any further assistance, please call us at (800) 930-5450. Following the receipt of this final report, a final invoice indicating the remaining payment will be sent to you. We will safely and securely dispose of all samples and confidential information in our possession in 30 days, unless otherwise instructed by your company. It has been a pleasure working with you and we look forward to serving you again. Sincerely, Avomeen Analytical Services

Craig D. Larner
Craig D. Larner, Ph.D. Technical Director 800-930-5450 craig@avomeen.com

Shri Thanedar
Shri Thanedar, Ph.D. CEO/Chief Chemist 800-930-5450 sthanedar@avomeen.com

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