Phenylethylamine and Hordenine Determination by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to describe a method for the quantitative analysis and
 identification of 2-phenylethylamine and hordenine in finished products and raw materials using
 
 HPLC and UV/VIS spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of 2-phenylethylamine and
 
 hordenine. This procedure is valid for 2-phenylethylamine and hordenine. Some excipients and
 
 dietary ingredients used in the finished products can interfere with the analysis of 2-
 Phenylethylamine. Other wavelengths can be used if interferences are present.
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC and Analytical Chemists to follow this procedure.
 
 3.2 It is the responsibility of QC Laboratory Management to implement this procedure and
 to ensure that the procedure is being followed. 
 
 3.3. It is the responsibility of QC Laboratory Management to keep this procedure current with
 
 latest Ion Labs practices. 
 
 4.0 Definitions 
 
 4.1 ACN- Acetonitrile 
 
 4.2 HeO - Deionized water 
 
 4.3. HCl-— Hydrochloride 
 
 4.4 2-PEA — 2-Phenylethylamine 
 
 4.5 QC-—Quality Control 
 
 4.6 CofA -— Certificate of Analysis 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Phenylethylamine and Hordenine Determination by D-745 4 Page 2 of 8 
 
 HPLC coupled with UV/VIS Spectroscopy 
 
 5.0 References 
 
 5.1 MV-LAB-14-012, Protocol, Open Validation Protocol for Amino Acids and Amines
 
 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Reagents: all reagents are HPLC grade or better. 
 
 6.1.1 Deionized water 
 
 6.1.2 ACN 
 
 6.1.3 Ammonium Acetate 
 
 6.1.4 2-phenylethylamine and/or hordenine 
 
 6.2. Supplies and Glassware 
 
 6.2.1 HPLC vials, 12mm X 32mm with screw cap enclosures w/ septa 
 
 6.2.2. 1L mobile phase container 
 
 6.2.3. 50mL, 100mL, and 500mL volumetric flasks 
 
 6.2.4 50mL and 100mL beakers 
 
 6.2.5 200uL, lmL, and 10mL pipette tips 
 
 6.2.6 1.5mL and 2.0mL micro centrifuge tubes 
 
 6.2.7 10mL plastic luer lock syringe 
 
 6.2.8 0.2 or 0.45um 25mm Nylon syringe filters 
 
 6.2.9 22mL screw cap vials (scintillation) 
 
 6.2.10 Weigh boats 
 
 6.3 Equipment 
 
 6.3.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 
 and UV detector with a chromatographic data handling system 
 
 6.3.2 Analytical Balance 
 
 6.3.3. Vortex 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Phenylethylamine and Hordenine Determination by D-745 Page 3 of 8 
 
 HPLC coupled with UV/VIS Spectroscopy 
 
 6.3.4 Stir Plate 
 
 6.3.5 Wrist action shaker 
 
 6.3.6 Micro-centrifuge 
 
 6.3.7 200uL, ImL and 10mL pipettes 
 
 7.0 Procedure 
 
 7.1 Mobile Phase A - 20mM Ammonium acetate (aq) 
 
 7.1.1. Dissolve 1.54 g of ammonium acetate in 1000 mL H20. Scale as necessary.
 
 7.2 | Mobile Phase B (ACN) - Acetonitrile 
 
 7.3. Diluent - Mobile Phase A 
 
 7.4 Standard Preparation 
 
 7.4.1. Use the actual purity from the CofA for 2-Phenylethylamine/hordenine in your
 calculations. All Standards are prepared by weighing no less than the minimum
 
 weight of the analytical balance. 
 
 7.4.2 Dissolve standard in two-thirds its final volume in an appropriately sized
 volumetric flask using Diluent. Mix on the wrist action shaker for 30 minutes then
 
 inspect to ensure complete dissolution. Once standard is fully dissolved, bring
 
 standard to final volume before using. 
 
 7.4.3 Dilutions are prepared using diluent. Dilutions can be made using volumetric
 flasks or using 10mL, ImL and 200uL variable pipettes. Specific standard
 
 concentrations will approximate the concentration expected to be found in the
 product being tested based on the sample dilution and calculated from the label.
 
 Dilutions can be prepared directly in HPLC vials. 
 
 7.5 Sample Preparation 
 
 7.5.1. The Sample Preparation must be within the linear range of the method:
 
 7.5.1.1 2-Phenethylamine — 0.006 mg/mL — 0.4 mg/mL 
 
 7.5.1.2 Hordenine — 0.01 mg/mL — 0.5 mg/mL 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Phenylethylamine and Hordenine Determination by D-745 4 Page 4 of 8 
 
 HPLC coupled with UV/VIS Spectroscopy 
 
 Tad 10 or more dosage units can be pooled and ground by mortar and pestle as
 
 necessary. 
 
 133 Based on the fill weight per dose weigh a portion of the pooled dosages to
 generate an analyte concentration that is within the validated linearity and
 
 solubility range for the analyte being tested. 
 
 7.5.4 Samples can be dissolved in diluent at any volume starting from 10mL. To
 manage large volumes the sample can be initially dissolved in a smaller volume
 
 that is within the solubility range and a portion further diluted to bring the analyte
 
 concentration into the linear range of measurement. 
 
 Taw The final diluted sample must be filtered or centrifuged before analyzing by
 HPLC. Filter through a 0.45 um membrane, discarding at least 0.5 mL before
 
 collecting a portion for analysis. From the collected sample dilute as needed then
 add ImL to an HPLC vial for analysis. 
 
 7.5.5.1 Alternatively centrifugation using the final diluted sample can be
 
 performed by filling an even number of 1.5mL or 2.0mL micro
 centrifuge tubes, then pellet out insoluble matter for 3 minutes at
 
 6000rpm. 
 
 For finished products or raw materials being analyzed for the first time using this
 128 
 method an in process validation is required to demonstrate spectral purity at the
 detection wavelength and extraction efficiency before quantitation can be
 
 completed. 
 
 7.6 Test Conditions 
 
 7.6.1 Gradient-Isocratic 
 
 Time %A %B Gradient type 
 
 0.00 85 15 0 
 
 10.00 85 15 0 
 
 7.6.2 Column- Luna C18(2), 5, 120A, 4.6 X 250mm 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Phenylethylamine and Hordenine Determination by D-745 
 Page 5 of 8 
 HPLC coupled with UV/VIS Spectroscopy 
 
 7.6.3 Flow Rate- 1.0mL/min 
 
 7.6.4 UV detection- 210nm 
 
 165 Injection volume- 20uL 
 
 7.6.6 Column Temperature- 40°C 
 
 7.6.7 Recommended 3-D Spectral Range- 200nm to 700nm 
 
 7.7 Recommended Sequence 
 
 beteh Make at least 2 injections of a Blank (Diluent). 
 
 TAD Make five injections of the Working Standard. 
 
 his Make a single injection of each Sample Preparation. 
 
 7.7.4 Make a single injection of the Working Standard after every six samples and at
 
 the end of the run. 
 
 7.8 System Suitability 
 
 7.8.1 The %RSD of five consecutive injections of the Working Standard is NMT 5.0%.
 
 7.8.2 The %RSD of all standard injections is NMT 5%. 
 
 7.8.3 Non-conforming results may trigger execution of a product specific method
 optimization per D-126/D-103 
 
 7.9 Column Wash and Storage 
 
 7.9.1 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 30 min.
 
 TIA Store the column with H2O / ACN (50/50). 
 
 7.10 Example calculations for determining finished product % label or raw material % purity
 
 7.10.1 % assay = — x aWtsiqxP x. =V P xSS x 100 
 Vstd SA LA 
 Ss 
 Ruy Sample peak area 
 
 Rg Mean standard peak area 
 
 Wtz.q Weight of reference standard in mg 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Phenylethylamine and Hordenine Determination by D-745 4 Page 6 of 8 
 
 HPLC coupled with UV/VIS Spectroscopy 
 
 V sta Volume of the standard preparation accounting for dilutions in mL
 
 P Purity of the reference standard in decimal format 
 
 SA Sample amount in mg (solids) or mL (liquids) 
 
 | Vspl Volume of the sample preparation accounting for dilutions in mL
 
 SS Serving size: Weight of a single dosage unit in mg for tablets and
 
 capsules, volume of a single serving from the theoretical formula in mL
 
 for liquids, or 1 for raw materials. 
 
 LA Label amount in mg per dose or | for raw materials 
 
 7.11 Example Chromatography 
 
 7.11.1 Blank 
 
 blank 
 350- 
 300- 
 250- 
 Z 200- 
 150- 
 100- 
 50- 
331.8441 NS

 05 1 15 #2 25 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 
 Time [min] 
 7.11.2 Working Standard (2-phenethylamine) 
 
 19AS111 0.1mg/ml 
 350- ae < 
 , UU 
 300+ 
 250+ 
 > 200- 
 150- os 
 "50,‘i M2m S~N M S o R r E n R n E a E n E n od © = 3 m 8 nN 
 0 >2 :¥ Lj SN— T Ni ' t t ? t ia Lj ' Lj ~ = UJ *> tT tj 
 0.5 1 1.5 2 2.5 2 oo 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 
 Time [min] 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Phenylethylamine and Hordenine Determination by D-745 4 Page 7 of 8 
 
 HPLC coupled with UV/VIS Spectroscopy 
 
 7.11.3 Sample (2-phenethylamine) 
 
 266CA1 0.1mg/mi 
 350- 
 300- 
 250- 
 4 200- 
 150+ 
 100- 
 50- 
 @) 
 051. 
 1+ 
 755.1 822.2 582.2 706.2 
 266.54ht 060,641 
 05 1 15 2 25 45 5 5&5. 
 Time [min] 
 7.11.1 Working Standard (hordenine) 
 ‘9AS050 0.1mg/ml 
 600 
 500- 
 400: 
 < 300- 
 200+ 
 100- 
 600 
 2eH+oN 
 121.24 472.2i 334.2 527.2 
863.34 gninedroh

 541.44 994.4#1 
 4 =DC 5.5 6.5 f > Oo Oo 3.5 4
 Time [min] 
 N nw ww 
 7.11.2 Sample (hordenine) 
 190042 0.1mg/ml 
 043 926. 
 3000- 
 2500- 
 -2000° 
 3500- 
 1000+ © 
 $0) 
 500- 0 
 ¥ 
 5 
 4a = 
 « 
 4 
896.3 eninedroh

 681.4 404.4\ 
 | 
00.5 .no

 781.5 543.5 955.54 
 ]ro 
 967.5 798.54 al 
 Time [min] 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Phenylethylamine and Hordenine Determination by D-745 4 Page 8 of 8 
 
 HPLC coupled with UV/VIS Spectroscopy 
 
 8.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 1 | 01/06/15 | New 01/14/19 Scheduled review: addition of hordenine to valid analytes, Updated 19-0040 J Meigen format to match current Ion practices Update to reflect current practices. Simplify mobile phase preparation. Add recommended sequence section. Replace | 15-0008 | X. Shao |
| 3 | 04/15/22 | requirements section with system suitability. Update example CC- calculation for consistency with current methods. Add example chromatography. | 22-0180 | S. Sassman |
| 4 | 12/05/22 | Minor edits. Added “recommended” to spectral range. CC- | 22-0459 | J. Sassman |