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Bioluminex Test Report
Iceberg lettuce - Diphenylamine Report Number: CDXA-BLR-058-00 Project Number: BIO001 Issue Date: September 08, 2005 Page: 1 of 8

1. OBJECTIVE Perform a Bioluminex fingerprint analysis on diphenylamine spiked and unspiked iceberg lettuce extracts.

2. SUMMARY Iceberg lettuce was freeze-dried, extracted with methanol and the extract spiked with diphenylamine and compared to the unspiked extract by HPTLC and Bioluminex analysis.

3. STANDARDS 3.1 3.2 3.3 Standards: Diphenylamine –Lot# 5028X, CDXA-05-0506 (023) Positive Control: Batch # 023K0054 Negative Control: ChromaDex, Lot# 20621-250

4. SAMPLES 4.1 Sample: Iceberg lettuce – CDXA-05-0484 (020)

5. REAGENTS 5.1 5.2 5.3 5.4 5.5 Methanol (HPLC Grade), EMD Lot #44317 Milli-Q Water Acetonitrile, 99.8%, EMD, Lot # 45178 Ethyl acetate, 98 %, Sigma-Aldrich, Batch # 00956DO Formic Acid, 98 %, Fluka, Lot # 455768/1

This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce – Diphenylamine CDXA-BLR-057-00 5.6 5.7 5.8 5.9 5.10 Page 2 of 8

Toluene, HPLC grade, 99.8%, Sigma-Aldrich, Batch # 01546EC Bioluminex Buffer A, Lot # M3711316 Vibrio fisheri, Lot # 02700-102 (L200705-1) Dry ice Tech methanol, Univar, Lot # DV045829070

6. EQUIPMENT 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.11 6.12 6.13 6.14 6.15 6.16 6.17 6.18 Mettler-Toledo AX205 Analytical Balance Coffee grinder 25 mL scintillation vials 0.45 um PTFE syringe filter Various laboratory glassware Aquasonic 50T Ultrasonic bath Camag automatic TLC sampler 4 (ATS-4) Barnstead Lab-Line mechanical oven Spray reagent flask Ridged-bottom TLC developing chamber Forma Scientific incubator/orbital shaker Camag automatic immersion device Alpha Innotech Fluorchem® 8900 Squeegee Corning stirrer/ hot plate BrandTech Scientific Accu-jet Pipetter Forma Scientific Biological Safety Cabinet Class IIA/B3 Labconco Lyph-Lock 18 Lyophilizer

7. STANDARD PREPARATION 7.1. A 1 mg/mL stock standard of diphenylamine was prepared by dissolving 5.80 mg of the standard into 5.80 mL acetonitrile. A 0.1 mg/mL analytical standard was prepared by diluting 0.5 mL of the stock standard to 5 mL using acetonitrile. An aliquot of the analytical standard was transferred to an amber HPLC vial for analysis. A 0.5 mg/mL solution of negative control was prepared by dissolving 5.31 mg of negative control into 10.62 mL Milli-Q water. The solution was sonicated for 5 minutes before transferring an aliquot to an amber HPLC vial for analysis. A 1.0 mg/mL solution of was prepared by dissolving 14.57 mg of negative control into 14.57 mL methanol (HPLC grade). The solution was sonicated for 5 minutes before transferring an aliquot to an amber HPLC vial for analysis.

7.2.

7.3.

This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce – Diphenylamine CDXA-BLR-057-00 8. SAMPLE PREPARATION/EXTRACTION A head of iceberg lettuce was chopped using a coffee grinder, transferred to a tared lyophilizer jar, and the gross wet weight determined. The sample was shell frozen using a dry ice/methanol bath, and then freezedried using the lyophilizer for approximately 118 hours. The final dry weight of the dried material and flask was measured. The net wet weight and dry weight were calculated and then used to determine the percent loss on drying (see table 1). Table 1. Loss on drying determination Sample # Tare Gross Wet Wt CDXA-034-178.2 504.7 g 869.2 g Page 3 of 8

Gross Dry Wt 523.20 g

Wet Wt 364.5 g

Dry Wt 18.5 g

%LOD 94.92

The dried material was ground using a mortar and pestle to homogenize the sample. Extracts were prepared by combining approximately 1 g of the sample (see table 2 for exact weights) with 10 mL methanol in separate 20 mL scintillation vials. Table 2. Sample extraction weights and volumes Sample Name Sample Methanol Amount added CDXA-036-004.1 (extraction #1) 1.1053 g 10 mL CDXA-036-004.2 (extraction #2) 1.1023 g 10 mL The solutions were sonicated for 15 minutes and placed in an oven at 65° for 1 hour under a sealed environment. Extracts were cooled to room temperature before removing the supernatants. The supernatants were then filtered using a Phenex 33 mm non-sterile 0.45 um PTFE syringe filter. The samples were then brought up to 10 mL using methanol in a 10 ml volumetric flask and residue analysis performed (see section 9 for details). Based on residue analysis extraction 2 was chosen for BioLuminex analysis. A 1 mL aliquot of extraction 2 was transferred to an amber HPLC vials for analysis. A spiked sample was prepared by adding a total of 40 µL of the stock diphenylamine standard (1 mg/mL) to 2 mL of extraction 2. An aliquot of the spiked sample was transferred to an amber HPLC vials for analysis.

9. SAMPLE RESIDUE Two residues for each extraction were performed. Four pre-dried aluminum weigh pans stored in a 100 o C oven were removed and cooled to room temperature before tare weights were recorded. Then using a
This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce – Diphenylamine CDXA-BLR-057-00 Page 4 of 8

calibrated automatic pipette (Gilson), 2 x 1.00 mL of each extraction was placed into each aluminum pan. The pans were gently heated using a hot plate (Corning Stirrer/Hot Plate) to evaporate the majority of solvent before placing pans in a mechanical oven at 100o C for 1 hour. After cooling the pan was again weighed to obtain a final weight. Table 3: Residue Weights Sample ID Volume (mL) Extraction 1, Residue 1 1.0 Extraction 1, Residue 2 1.0 Extraction 2, Residue 1 Extraction 2, Residue 2 1.0 1.0

Tare (g) 1.2546 1.2588 1.2617 1.2626

Final (g) 1.2783 1.2831 Average 1.2862 1.2868 Average

Residue (mg/mL) 23.70 24.30 24.00 24.50 24.20 24.35

10. ANALYSIS METHOD 10.1 Chromatography: The samples and standards were applied in methanol to a pre-washed (methanol elution) 10 x 10 cm Merck HPLTC silica gel 60 F254 plates (Lot # OB494661 ) via a CAMAG Automatic TLC Sampler 4 (ATS-4) at y = 8 mm using band spray application set to an ethanol application mode. After sample application the plates were to air dried for 15 minutes. The plates were developed to 70 mm using a mobile phase of toluene: ethyl acetate: formic acid: water (7:5:1.1:0.2) in a pre-equilibrated (30 minutes) 10 x 10 cm ridged bottom chamber. Post development the plate was dried in a mechanical oven at 40 °C for 2 hours. 10.2 Data Image 1: After plate drying a UV image at 254 nm was recorded and collected with the Alpha Innotech Fluorchem 8900 imaging system. 10.3 Data Image 2: After plate drying a UV image at 365 nm was recorded and collected with the Alpha Innotech Fluorchem 8900 imaging system. 10.4 Data Image 3: After UV image recordings the plate was immersed into 190 mL of acid compatible (Buffer A) cultured BioLuminex bacteria using a CAMAG
This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce – Diphenylamine CDXA-BLR-057-00 Page 5 of 8

automatic immersion device set a speed of 1 with no holding. The excess bacteria was removed from the plate using a squeegee and the results recorded (Alpha Innotech Fluorchem® 8900 imaging system) as five images, each with a 2 minute exposure time over a total time of 10 minutes.

This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce – Diphenylamine CDXA-BLR-057-00 11. RESULTS & CHROMATOGRAMS UV 365 nm Page 6 of 8

UV 254 nm

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Yellow line = sample origin Red line = solvent front @70mm

Lane Sample Application Volume Analyte Amount 1 Lettuce – Diphenylamine 0.5 µL 10 ng* 2 Lettuce – Diphenylamine 1.0 µL 20 ng* 3 Lettuce – Diphenylamine 3.0 µL 60 ng* 4 Lettuce 0.5 µL N/A 5 Lettuce 1.0 µL N/A 6 Lettuce 3.0 µL N/A 7 (blank) 8 Diphenylamine 0.5 µL 50 ng 9 Negative Control 4.0 µL 4 µg 10 Positive Control 2.0 µL 8 µg *Assuming 100% recovery of the analyte. Actual recovery has not been determined.

This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce - Diphenylamine CDXA-BRM-058-00 Page 7 of 8

Bioluminex

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Yellow line = sample origin Red line = solvent front @70mm Lane Sample Application Volume Analyte Amount 1 Lettuce – Diphenylamine 0.5 µL 10 ng* 2 Lettuce – Diphenylamine 1.0 µL 20 ng* 3 Lettuce – Diphenylamine 3.0 µL 60 ng* 4 Lettuce 0.5 µL N/A 5 Lettuce 1.0 µL N/A 6 Lettuce 3.0 µL N/A 7 (blank) 8 Diphenylamine 0.5 µL 50 ng 9 Negative Control 4.0 µL 4 µg 10 Positive Control 2.0 µL 8 µg *Assuming 100% recovery of the analyte. Actual recovery has not been determined.

This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.

Iceberg lettuce – Diphenylamine CDXA-BLR-057-00 12. REFERENCES 11.1 11.2 11.3 11.4 11.5 13. ChromaDex Analytics notebook CDXA-035, p 102, 127, 139, 151 ChromaDex Analytics notebook CDXA-036, p 4-5 ChromaDex Analytics notebook CDXA-034, p 178, 182 Bioluminex Instruction Manual – Standard Kit ChromaDex Method CDXA-BLM-033-00 Page 8 of 8

SIGNATURES Author: Gerald T Gourdin Verified: Sheryl Verbitski

This document has a digital signature on the last page. The digital signature must be verified for this document to be authentic. This product analysis is subject to our General Terms and Conditions of Business, including our services and reports, and is for the benefit of our client only and may not be relied upon by any other party and relates solely to the sample(s) in our possession and cannot be applied to any other material.


				
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