Confidential Report - Lost Empire Herbs · 2020. 1. 27. · Chromatography Parameters . Instrument: Agilent 1100 Series HPLCconnected to AB Sciex API 2000 MS, ID: R149, Calibration

Confidential Report

«Lost Empire Herbs»

Issue Date: October 21, 2019 Project Code: 19-70311

Project Code No. 19-70311

FDA Registered | cGMP Compliant Facility |DEA Licensed Avomeen Analytical Services | 4840 Venture Drive, Ann Arbor, MI 48108 Services | www.avomeen.com |800-930-5450

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Logan Christopher Lost Empire Herbs United States (831)-419-4770 [email protected]

Description of Project Thank you for contacting Avomeen Analytical Services to identify by LC-MS-MS, if the following controlled substances: Testosterone (TS), Epitestosterone (ET), Androstenedione (AD), Dehydroepiandrosterone (DHEA) and Androsterone (AS) are present in pine pollen powder provided by Lost Empire Herbs. Following are the results, methodology, and data associated with our analysis of the samples.

Table 1: Sample Description

Avomeen Sample ID Sample Description

26SEP19LO2088 Megadose Pine Pollen 250g, black bag, yellow dusty powder

Executive Summary The primary goal of this study was to detect and quantitate if present, five steroids (Testosterone (TS), Epitestosterone (ET), Androstenedione (AD), Dehydroepiandrosterone (DHEA) and Androsterone (AS) in Pine pollen powder by a LC-MS-MS method. Three separate LC-MS-MS methods were developed: A Multiple reaction monitoring (MRM) method for TS, ET & AD, and two separate Selective Ion Monitoring (SIM) methods for DHEA and AS respectively. Calibration curves were built for all of the five steroids over different concentration ranges. A concentrated sample of Pine pollen powder provided by Lost Empire Herbs was prepared and analyzed for presence of TS, ET, AD, DHEA & AS. The amount of steroids present were calculated by comparing both HPLC-UV & MS spectrums of the steroids with that of the Pine pollen powder using their respective extraction ions. Table 2 shows the summary of the results obtained.

Table 2. Test results for Steroids (TS, ET, AD, DHEA & AS) in Pine pollen powder samples

Analytes Amount present in Pine Pollen powder Limit of Detection (ppb) Testosterone (TS) None detected 10

Epitestosterone (ET) None detected 10 Androstenedione (AD) None detected 10

Dehydroepiandresterone (DHEA) None detected 500 Androsterone (AS) None detected 500

Quality Statement The work reported herein was conducted non-GMP and was not reviewed by Quality Assurance. All data in this report accurately reflects the raw data stored in the archives of Avomeen Analytical Services.

Analytical Testing Initial Observations A photograph of the samples “As Received” can be found in Figure 1.



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Figure 1: Photograph of the Samples “As Received”

Sample and Solution Preparation for the LC/MS/MS analysis: Diluent: Methanol Mobile Phases: Mobile Phase A: 0.1% Formic acid in Purified Water: 1L of purified water was transferred into 1-L mobile phase bottle. 1 mL of formic acid was pipetted into the bottle. The solution was mixed well and sonicated to degas. Mobile Phase B: Acetonitrile (ACN): 1L of ACN was transferred into 1-L mobile phase bottle. The solution was sonicated to degas. Sample preparation: Sample (100,000 ppm): 1.17165 g of Pine pollen powder sample was weighed and transferred into 10-mL volumetric flask (VF). Diluted to volume (Q.S.) with diluent and mixed well by sonication for 20 min. The solution was filtered using 0.45 µm PTFE syringe filter. Methods of Analysis: 3 different LC-MS methods were used for analysis of 5 steroids in pine pollen powder. Testosterone (TS), Epitestosterone (ET) and Androstenedione (AD) were analyzed together by a single LC-MS method while two separate LC-MS methods were used for analysis of Dehydroepiandrosterone (DHEA) and Androsterone (AS) individually. Testosterone (TS), Epitestosterone (ET) and Androstenedione (AD) Analysis: TS Stock Standard (1000 ppm): 9.55 mg of Testosterone (USP reference standard) was transferred into 10-mL VF. Diluted to volume (Q.S.) with diluent and mixed well. ET Stock Standard (1000 ppm): ET vial containing 1 mg/mL of ET in 1 mL ACN obtained from Cerilliant was used as reference standard. AD Stock Standard (1000 ppm): AD vial containing 1 mg/mL of AD in 1 mL ACN obtained from Cerilliant was used as reference standard. TS+ET+AD standard (10 ppm): 0.1 mL of TS Stock Standard (1000 ppm), 0.1mL of ET Stock Standard (1000 ppm) and 0.1 mL of AD Stock Standard (1000 ppm) was pipetted into 10-mL VF. Q.S. with diluent and mixed well. TS+ET+AD standard (1 ppm): 1 mL of the TS+ET+AD standard (10 ppm) solution was pipetted into 10-mL VF. Q.S. with diluent and mixed well. Linearity Standards: Linearity standards were prepared in range 20 ppb to 100 ppb for all three steroids: TS, ET and AD. All linearity standards were prepared using TS+ET+AD standard (1 ppm). Preparation details are shown in Table 3.

Table 3: Linearity standards for TS, ET & AD

TS concentration (ppb) ET concentration (ppb) AD concentration (ppb) Vol. of TS+ET+AD Std (1 ppm) Total vol. with diluent 19.10 20.00 20.00 0.2 10 38.20 40.00 40.00 0.4 10



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57.30 60.00 60.00 0.6 10 76.40 80.00 80.00 0.8 10 95.50 100.00 100.00 1.0 10

TS+ET+AD Limit of Detection (LOD) solution (10 ppb): 0.1 mL of TS+ET+AD Standard (1 ppm) was pipetted into 10-mL VF. Q.S. with diluent and mixed well. Instrument Conditions for each type of analysis. Chromatography Parameters Instrument: Agilent 1100 Series HPLC connected to AB Sciex API 2000 MS, ID: R149, Calibration due: 31 Dec 2019 Column: XBridge C18, 100 mm x 2.1 mm, 3.5 µm, part # 186003022, SN: 01793612714044 Flow rate: 0.3 mL/min Mobile Phase A: 0.1% Formic acid in Purified Water Mobile Phase B: Acetonitrile (ACN) Inj. Vol.: 20 µL Run time: 20 min Column Temp: 30˚ C Sample Temp: Not controlled Elution: Gradient

Table 4. Mobile phase gradient Time (min) MP A (%) MP B (%)

0.00 99 1 12.00 1 99 15.00 1 99 15.10 99 1 20.00 99 1

Mass Spectrometry Parameters Detection Mode: ESI positive, Multiple Reaction Monitor (MRM) Curtain Gas: 14.00 IonSpray Voltage: 4500.00 Temperature: 550.00 Collision Gas: 10.00 Ion Source Gas I: 20.00 Ion Source Gas II: 20.00 Dwell time (msec): 250.00

Table 5: MRM parameters

Analyte Ion Pair (Da) DP (Volts)

FP (Volts)

EP (Volts)

CE (Volts)

CXP (Volts)

Testosterone 289.2/97.0 30 200 10 40 15 Epitestosterone 289.0/109.0 30 200 10 40 15

Androstenedione 287.2/97.1 30 200 10 40 15 *DP: Declustering potential; FP: Focusing Potential; EP: Entrance Potential; CE: Collision Energy; CXP: Collision Cell Exit Potential

Results and Discussion Note: TS & ET being stereoisomers of each other showed same molecular ion and fragmentation ion peaks hence the HPLC was used to distinguish between TS peak (RT: ~ 15 min) and ET peak (RT: ~ 16) using retention time. Diluent: Diluent showed no peaks at the retention time of TS, ET & AD. Diluent chromatograms for TS, ET & AD are shown in figures 2, 3 & 4 respectively. Limit of Detection (LOD) solution: LOD solution, TS+ET+AD mixture containing 10 ppb of each analyte showed Signal to Noise (S/N) ratio above 3 for each of the analyte. This was the minimum amount of steroids: TS, ET and AD that were possible to detect if present in pollen powder.



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Table 6 shows the concentration and S/N of TS, ET and AD in the LOD solution. Chromatograms showing S/N ratios for TS, ET and AD are shown in figures 5, 6 and 7 respectively.

Table 6: Signal to Noise ratio for TS, ET and AD in TS+ET+AD LOD solution Analyte LOD Conc. (ppb) Signal to Noise ratio (S/N)

Testosterone (TS) 9.55 6.9 Epitestosterone (ET) 10.00 27.5

Androstenedione (AD) 10.00 13.2 Linearity: Linearity of the response for TS, ET and AD was determined by analyzing TS+ET+AD linearity standards over a range from 20 ppb - 100 ppb. The area responses of the TS, ET & AD in the standards were plotted against the concentrations of TS, ET & AD respectively in the standard solutions. Correlation coefficient of 1.00, 1.00 & 0.99 were obtained for TS, ET & AD respectively demonstrating that the peak areas for TS, ET & AD were linear over the chosen range. The results for TS, ET & AD are presented in Table 7, 8 & 9 respectively. The plot of the linearity standards for TS, ET & AD are shown in Figures 8, 9 & 10 and chromatograms for linearity standards can be seen in Figures 11, 12 & 13 respectively.

Table 7: Linearity for TS TS Linearity Standards Concentration (ppb) Area (Counts)

19.10 3750 38.20 8210 57.30 12300 76.40 15700 95.50 19200 Slope 200.99

Intercept 315.00 Correlation Coefficient (r) 1.00

Table 8: Linearity for ET

ET Linearity Standards Concentration (ppb) Area (Counts) 20.00 4360 40.00 8440 60.00 12400 80.00 16300

100.00 20300 Slope 198.70


Table 9: Linearity for AD

ET Linearity Standards Concentration (ppb) Area (Counts) 20.00 3110 40.00 5930 60.00 8110 80.00 10900

100.00 15300 Slope 146.75

Intercept -135.00 Correlation Coefficient (r) 0.99

Sample Analysis: Pine pollen powder sample (100,000 ppm) was analyzed and checked for the presence of steroids, TS, ET and AD by comparing both the HPLC-UV and MS spectrums with the linearity standards. No match with both HPLC-UV and MS spectrums confirmed TS, ET and AD were not present in pine pollen powder samples. HPLC-UV and MS comparison spectrums for TS (100 ppb) and pine powder (Figure 14 & 15), ET (100 ppb) and pine powder (Figure 16 & 17), AD (100 ppb) and pine powder (Figure 18 & 19) are shown below.



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Figures

Figure 2. Representative chromatogram for TS diluent

Figure 3. Representative chromatogram for ET diluent



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Figure 4. Representative chromatogram for AD diluent

Figure 5. Representative chromatogram for TS LOD (10 ppb)



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Figure 6. Representative chromatogram for ET LOD (10 ppb)

Figure 7. Representative chromatogram for AD LOD (10 ppb)



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Figure 8. Linearity plot for TS

Figure 9. Linearity plot for ET

y = 200.99x + 315R² = 0.9964

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y = 198.7x + 438R² = 0.9999

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Figure 10. Linearity plot for AD

Figure 11. Chromatograms for TS linearity standards from 20 ppb – 100 ppb

y = 146.75x - 135R² = 0.9826

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Figure 12. Chromatograms for ET linearity standards from 20 ppb – 100 ppb

Figure 13. Chromatograms for AD linearity standards from 20 ppb – 100 ppb



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Figure 14. Representative HPLC-UV & MS Chromatogram for TS linearity standards 100 ppb

Figure 15. Representative HPLC-UV & MS Chromatogram for pine pollen powder sample using TS extraction ions



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Figure 16. Representative HPLC-UV & MS Chromatogram for ET linearity standards 100 ppb

Figure 17. Representative HPLC-UV & MS Chromatogram for pine pollen powder sample using ET extraction ions



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Figure 18. Representative HPLC-UV & MS Chromatogram for AD linearity standards 100 ppb

Figure 19. Representative HPLC-UV & MS Chromatogram for pine pollen powder sample using AD extraction ions



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Dehydroepiandreosterone (DHEA) Analysis: DHEA Stock Standard I (1000 ppm): DHEA vial containing 1 mg/mL of ET in 1 mL methanol obtained from Cerilliant was used as reference standard. DHEA Stock Standard II (100 ppm): 1 mL of DHEA Stock Standard I (1000 ppm) was pipetted into 10-mL VF. Q.S. with methanol and mixed well. Linearity Standards: Linearity standards were prepared in range 1 ppm to 10 ppm for DHEA. All linearity standards were prepared using DHEA Stock Standard II (100 ppm). Preparation details are shown in Table 10.

Table 10: Linearity standards for DHEA

DHEA concentration (ppm) Vol. of DHEA Stock Std II (100 ppm) Total vol. with diluent 1.00 0.1 10 2.00 0.2 10 4.00 0.4 10 6.00 0.6 10 8.00 0.8 10

10.00 1.0 10 DHEA Limit of Detection (LOD) solution (0.5 ppm): 0.05 mL of DHEA Stock Standard (100 ppm) was pipetted into 10-mL VF. Q.S. with diluent and mixed well. Instrument Conditions for each type of analysis. Chromatography Parameters Instrument: Agilent 1100 Series HPLC connected to AB Sciex API 2000 MS, ID: R149, Calibration due: 31 Dec 2019 Column: XBridge C18, 100 mm x 2.1 mm, 3.5 µm, part # 186003022, SN: 01793612714044 Flow rate: 0.3 mL/min Mobile Phase A: 0.1% Formic acid in Purified Water Mobile Phase B: Acetonitrile (ACN) Inj. Vol.: 20 µL Run time: 20 min Column Temp: 30˚ C Sample Temp: Not controlled Elution: Gradient


0.00 99 1 12.00 1 99 15.00 1 99 15.10 99 1 20.00 99 1

Mass Spectrometry Parameters Detection Mode: ESI positive, Selective Ion Monitoring (SIM) Curtain Gas: 20.00 IonSpray Voltage: 4500.00 Temperature: 550.00 Collision Gas: 10.00 Ion Source Gas I: 20.00 Ion Source Gas II: 0.00 Dwell time (msec): 100.00



FP (Volts)

EP (Volts)

CEP (Volts)

Dehydroepiandreosterone 289.2 20 200 10 15.76 *DP: Declustering potential; FP: Focusing Potential; EP: Entrance Potential; CEP: Collision Energy Potential



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Results and Discussion Diluent: Diluent showed no peaks at the retention time of DHEA. Diluent chromatogram for DHEA is shown in figure 20. Limit of Detection (LOD) solution: LOD solution, DHEA (0.5 ppm) showed Signal to Noise (S/N) ratio above 3 for the analyte. This was the minimum amount of DHEA, this method is capable of detecting. Table 13 shows the concentration and S/N of DHEA in the LOD solution. Chromatogram showing S/N ratios for DHEA is shown in figure 21.

Table 13: Signal to Noise ratio for DHEA in DHEA LOD solution Analyte LOD Conc. (ppm) Signal to Noise ratio (S/N)

Dehydroepiandreosterone (DHEA) 0.50 4.6 Linearity: Linearity of the response for DHEA was determined by analyzing DHEA linearity standards over a range from 1 ppm - 10 ppm. The area responses of the DHEA in the standards were plotted against the concentrations of DHEA respectively in the standard solutions. Correlation coefficient of 1.00 was obtained for DHEA demonstrating that the peak areas for DHEA were linear over the chosen range. The results for DHEA are presented in Table 14. The plot of the linearity standards for DHEA is shown in Figure 22 and chromatograms for linearity standards can be seen in Figure 23.

Table 14: Linearity for DHEA DHEA Linearity Standards Concentration (ppm) Area (Counts)

1.00 66900 2.00 145000 4.00 268000 6.00 443000 8.00 508000

10.00 680000 Slope 66486.30


Sample Analysis: Pine pollen powder sample (100,000 ppm) was analyzed and checked for the presence of DHEA by comparing both the HPLC-UV and MS spectrums with the linearity standards. No match with both HPLC-UV and MS spectrums confirmed the absence of DHEA in pine pollen powder samples. HPLC-UV and MS comparison spectrums for DHEA (10 ppm) and pine powder (Figure 24 & 25) are shown below.



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Figures

Figure 20. Representative chromatogram for DHEA diluent

Figure 21. Representative chromatogram for DHEA LOD (0.5 ppm)



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Figure 22. Linearity plot for DHEA

Figure 23. Chromatogram for DHEA Linearity (1 ppm- 10 ppm)

y = 66486x + 8304.1R² = 0.9909

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Figure 24. Representative HPLC-UV & MS Chromatogram for DHEA linearity standards 10 ppm

Figure 25. Representative HPLC-UV & MS Chromatogram for pine pollen powder sample using DHEA extraction ions

Androsterone (AS) Analysis: AS Stock Standard I (1000 ppm): 5.37 mg of AS (Sigma Aldrich reference standard) was transferred into 5-mL VF. Q.S. with methanol and mixed well.



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AS Stock Standard II (100 ppm): 1 mL of AS Stock Standard I (1000 ppm) was pipetted into 10-mL VF. Q.S. with methanol and mixed well. Linearity Standards: Linearity standards were prepared in range 1 ppm to 8 ppm for AS. All linearity standards were prepared using AS Stock Standard II (100 ppm). Preparation details are shown in Table 15.

Table 15: Linearity standards for AS

AS concentration (ppm) Vol. of AS Stock Std II (100 ppm) Total vol. with diluent 1.07 0.1 10 2.15 0.2 10 4.30 0.4 10 6.44 0.6 10 8.59 0.8 10

AS Limit of Detection (LOD) solution (0.5 ppm): 0.05 mL of AS Stock Standard (100 ppm) was pipetted into 10-mL VF. Q.S. with diluent and mixed well. Instrument Conditions for each type of analysis. Chromatography Parameters Instrument: Agilent 1100 Series HPLC connected to AB Sciex API 2000 MS, ID: R149, Calibration due: 31 Dec 2019 Column: XBridge C18, 100 mm x 2.1 mm, 3.5 µm, part # 186003022, SN: 01793612714044 Flow rate: 0.3 mL/min Mobile Phase A: 0.1% Formic acid in Purified Water Mobile Phase B: Acetonitrile (ACN) Inj. Vol.: 20 µL Run time: 20 min Column Temp: 30˚ C Sample Temp: Not controlled Elution: Gradient


0.00 99 1 12.00 1 99 15.00 1 99 15.10 99 1 20.00 99 1

Mass Spectrometry Parameters Detection Mode: ESI positive, Selective Ion Monitoring (SIM) Curtain Gas: 20.00 IonSpray Voltage: 4500.00 Temperature: 550.00 Collision Gas: 10.00 Ion Source Gas I: 20.00 Ion Source Gas II: 0.00 Dwell time (msec): 100.00



FP (Volts)

EP (Volts)

CEP (Volts)

Androsterone 291.44 20 200 10 15.81 *DP: Declustering potential; FP: Focusing Potential; EP: Entrance Potential; CEP: Collision Energy Potential



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Results and Discussion Diluent: Diluent showed no peaks at the retention time of AS. Diluent chromatogram for AS is shown in figure 26. Limit of Detection (LOD) solution: LOD solution, AS (0.5 ppm) showed Signal to Noise (S/N) ratio above 3 for the analyte. This was the minimum amount of AS, this method is capable of detecting. Table 18 shows the concentration and S/N of AS in the LOD solution. Chromatogram showing S/N ratios for AS is shown in figure 27.

Table 18: Signal to Noise ratio for AS in AS LOD solution Analyte LOD Conc. (ppm) Signal to Noise ratio (S/N)

Androsterone (AS) 0.54 13.2 Linearity: Linearity of the response for AS was determined by analyzing AS linearity standards over a range from 1 ppm - 8 ppm. The area responses of the AS in the standards were plotted against the concentrations of AS respectively in the standard solutions. Correlation coefficient of 0.99 was obtained for AS demonstrating that the peak areas for AS were linear over the chosen range. The results for AS are presented in Table 19. The plot of the linearity standards for AS is shown in Figure 28 and chromatograms for linearity standards can be seen in Figure 29.

Table 19: Linearity for AS AS Linearity Standards Concentration (ppm) Area (Counts)

1.07 117000 2.15 242000 4.30 392000 6.44 527000 8.59 622000

Slope 66056.91 Intercept 82030.49

Correlation Coefficient (r) 0.99

Sample Analysis: Pine pollen powder sample (100,000 ppm) was analyzed and checked for the presence of AS by comparing both the HPLC-UV and MS spectrums with the linearity standards. No match with both HPLC-UV and MS spectrums confirmed the absence of AS in pine pollen powder samples. HPLC-UV and MS comparison spectrums for AS (10 ppm) and pine powder (Figure 30 & 31) are shown below.



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Figures

Figure 26. Representative chromatogram for AS diluent

Figure 27. Representative chromatogram for AS LOD (0.5 ppm)



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Figure 28. Linearity plot for AS

Figure 29. Chromatogram for AS Linearity (1 ppm- 8 ppm)

y = 66057x + 82030R² = 0.9796

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Figure 30. Representative HPLC-UV & MS Chromatogram for AS linearity standards 8 ppm

Figure 31. Representative HPLC-UV & MS Chromatogram for pine pollen powder sample using AS extraction ions



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Description of Instrumentation Used High Pressure Liquid Chromatography/Mass Spectroscopy (HPLC/MS): This method can be used for the analysis of samples that are less volatile, polar, and not thermally stable or consist of large molecules. Avomeen utilizes an Applied Biosystems API3000 triple-quad mass spectrometer coupled with an Agilent 1100 HPLC for LC/MS/MS analysis. The system is equipped with two different types of sources for the analysis of a great variety of chemicals. ESI (electrospray ionization) is used for polar compounds and large molecules and APCI (atmospheric pressure chemical ionization), a soft ionization technique, is used for the analysis of relatively non-polar compounds.

History

Date Revision Changes

21 OCT 19 .00 New document

Wrap Up Thank you for consulting with Avomeen Analytical Services. Results relate only to items tested. 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. Reports shall not be reproduced, except in full, without approval from Avomeen, LLC in writing. It has been a pleasure working with you and we look forward to serving you again. Sincerely,

Veeren Dewoolkar Sr. Analytical Chemist, Ph. D. [email protected]

Nimisha Patel Project Leader, M.S. [email protected]

Evan Boyst Sr. Technical Director, B.S. [email protected]

Andrew Kolbert Andrew C. Kolbert, Ph.D., M.T.M. President/Chief Technology Officer [email protected]

For questions or to arrange a teleconference, please contact your Project Manager, Kyle Martin, at 800-930-5450 or [email protected].

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Confidential Report - Lost Empire Herbs · 2020. 1. 27. · Chromatography Parameters . Instrument: Agilent 1100 Series HPLCconnected to AB Sciex API 2000 MS, ID: R149, Calibration