Raspberry Ketone and Zingerone Determination by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define a method for the quantitative analysis and/or
 
 identification of raspberry ketone (4-(4-hydroxyphenyl)-butanone) and zingerone
 (vanillylacetone) in raw materials and finished products using HPLC and UV/VIS
 
 spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of raspberry ketone and zingerone.
 
 Some excipients and dietary ingredients used in finished products may interfere with the analysis
 of raspberry ketone or zingerone. 
 
 3.0 Responsibility 
 
 3.1 It is the responsibility of QC 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 and Analytical Development Management to
 keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 Raspberry Ketone — 4-(4-hydroxyphenyl)-2-butanone 
 
 4.2 Zingerone — Vanillylacetone or 4-(4-hydroxy-3methoxyphenyl)-2-butanone
 4.3. UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.4 HsPQs4-— Phosphoric Acid 
 4.5 CofA — Certificate of Analysis 
 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rey 
 Raspberry Ketone and Zingerone Determination by D-727 6 Page 2 of 8 
 HPLC using UV/VIS Spectroscopy 
 
 4.6 H20 — Water 
 
 4.7 QC- Quality Control 
 
 5.0 References 
 
 5.1 MV-LAB-12-009, Protocol, Raspberry Ketone 4(4-Hydroxyphenyl)-2Butanone
 
 Determination by HPLC 
 
 5.2. MV-LAB-18-067, Protocol, Zingerone Determination by HPLC using UV/VIS
 Spectroscopy 
 
 6.0 Reagents, Supplies, Glassware and Equipment 
 
 6.1 Reagents: all reagents are HPLC or better. 
 
 6.1.1 H20 © 18.2 MQ-cm) 
 
 6.1.2 Acetonitrile 
 
 6.1.3. Methanol 
 
 6.1.4 H3PO, 
 
 6.1.5 Raspberry Ketone reference standard 
 
 6.1.6 Zingerone reference standard 
 
 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 10mL, 50mL, 100mL, 500mL, and 1L volumetric flasks 
 
 6.2.4 200uL, lmL, and 10mL pipette tips 
 
 6.2.5 10mL Plastic luer-lock syringes 
 
 6.2.6 0.45uM Nylon syringe filters 
 
 6.2.7 22mL screw cap vials 
 
 6.2.8 1.5mL and 2.0mL micro centrifuge tubes 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev 
 Raspberry Ketone and Zingerone Determination by Page 3 of 8 
 D-727 6 
 HPLC using UV/VIS Spectroscopy 
 6.2.9 Weigh paper and 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 Column - Sum, C5, 150mm X 4.6mm 
 
 6.3.3 Analytical Balance 
 
 6.3.4 Stir Plate 
 
 6.3.5 Wrist Action Shaker 
 
 6.3.6 Vortex 
 
 6.3.7 Sonicator Bath 
 
 6.3.8 200uL, ImL, and 10mL Pipettes- adjustable 
 
 7.0 Procedure 
 
 7.1 Mobile Phase Preparation 
 
 tolled Mobile Phase A (0.1% H3PO4 in H20) 
 
 7.1.1.1 Transfer 1000 mL of H20 to a 1000-mL mobile phase bottle.
 
 7.1.1.2 Add 1.0 mL H3PQs, and mix well. 
 
 V1.2 Mobile Phase B (0.1% H3PQOsg in acetonitrile) 
 
 7.1.2.1 Transfer 1000 mL of acetonitrile to a 1000-mL mobile phase bottle.
 
 7.1.2.2. Add 1.0 mL H3PQs, and mix well. 
 
 7.1.3 Diluent (100% methanol) 
 
 7.2 Standard Preparation 
 
 7.2.1 The linear range of the method is listed below. All standard and sample
 
 preparations must be within the linear range. 
 
 7.2.1.1. Raspberry Ketone — 0.0125 to 0.25 mg/mL 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev 
 Raspberry Ketone and Zingerone Determination by D-727 6 Page 4 of 8 
 HPLC using UV/VIS Spectroscopy 
 
 7.2.1.2 Zingerone — 0.01 to 0.1 mg/mL 
 
 7.2.2 Use the actual purity from the CofA for raspberry ketone or zingerone reference
 standard in your calculations. The Standard Preparation reflects 100% of the label
 
 quantity for raspberry ketone and zingerone. 
 
 7.2.3 All standards are prepared by weighing no less than the minimum weight of the
 analytical balance. Dissolve in and dilute to volume in an appropriately sized
 
 volumetric flask using Diluent. 
 
 7.2.4 Dilutions can be made using volumetric flasks or using ImL and 200uL variable
 
 pipettes. Working 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. Final dilutions may be prepared directly in HPLC vials.
 
 7.3. Sample Preparation 
 
 7.3.1 At least 20 dosage units are pooled and ground by mortar and pestle as necessary.
 
 7.3.2 Samples are prepared by weighing no less than the minimum weight of the
 
 analytical balance. 
 
 7.3.3. Samples can be dissolved in Diluent at any volume starting from 25mL. To
 manage large volumes the sample can be initially dissolved in a smaller volume
 
 and a portion further diluted to bring the analyte concentration into the linear
 range of measurement. 
 
 7.3.4 Based on the label claim and fill or tablet weight per dose for finished products
 
 or expected potency for raw materials, weigh a portion of the sample into a
 
 suitably sized volumetric flask to generate an analyte concentration that is within
 the validated linear range for the analyte being tested. 
 
 7.3.5 Dilute the sample to 2/3 of the flask volume with Diluent, and shake for 20
 
 minutes to facilitate dissolution. Sonication for 10 minutes can also be used to
 assist dissolution. Once the analyte is completely dissolved, bring sample up to
 
 volume with Diluent before any further dilutions. 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev 
 Raspberry Ketone and Zingerone Determination by D-727 6 Page 5 of 8 
 HPLC using UV/VIS Spectroscopy 
 
 7.3.6 If sonication is used, allow sample to cool to RT before continuing.
 
 7.3.7 For filtration, using the final large scale diluted sample withdraw up to 10mL
 using a 10mL plastic syringe then filter and discard at least 0.5mL of sample
 
 before collecting. From the collected sample dilute as needed then add 1mL to
 an HPLC vial for analysis. 
 
 7.3.8 For centrifugation using the final large scale diluted sample, fill an even number
 of 1.5 or 2.0mL micro-centrifuge tubes and pellet insoluble matter for 5 minutes
 
 at 6000rpm. 
 
 7.3.9 For finished products or raw materials being analyzed for the first time using this
 method an in process validation is required to demonstrate spectral purity,
 
 baseline separation of peaks and extraction efficiency as a part of system
 suitability before data can be reported using this method. 
 
 7.4 Test Conditions 
 
 TAI Gradient - Isocratic 65% Mobile Phase A : 35% Mobile Phase B 
 
 7.4.2 Column - 5um, C5, 100A, LC column, 150mm X 4.6mm 
 
 7.4.3 Flow Rate - 1.0 mL/min 
 
 7.4.4 UV Detection - 280nm 
 
 7.4.5 Injection Volume - 20uL 
 
 7.4.6 Temperature - 45°C 
 
 7.4.7 3-D Spectral Range- 200nm to 350nm 
 
 7.5 Recommended Sequence 
 
 7.5.1 Make at least 2 injections of a Blank (Diluent). 
 
 7.5.2 Make five injections of the Working Standard. 
 
 7.5.3 Make a single injection of each Sample Preparation. 
 
 

[SOP 

 Standard Operating Procedure 
 SOP No Rev 
 Raspberry Ketone and Zingerone Determination by Page 6 of 8 
 D-727 6 
 HPLC using UV/VIS Spectroscopy 
 7.5.4 Make a single injection of the Working Standard after every six samples and at
 the end of the run. 
 
 7.6 System Suitability 
 
 7.6.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.
 
 7.6.2. The %RSD of all Working Standard injections is NMT 5%. 
 
 7.7 Column Wash and Storage 
 
 7.7.1 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 15 min.
 
 7.7.2 Store the column with H2O / ACN (50/50). 
 
 8.0 Example Calculations 
 
 8.1 07 0 assay Y= — = — R ®, *u X Wt V sse tttdg a X P x .. V S sApl “x* L Ss A x 100
 
 Ry Sample peak area 
 
 Re Mean standard peak area 
 
 Wt.iqg Weight of reference standard in mg 
 
 Vstq Wolume 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) 
 
 Vp, | 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 1 for raw materials 
 
 

[SOP 

 Standard Operating Procedure Or 
 Raspberry Ketone and Zingerone Determination by eee > Page 7 of 8 
 HPLC using UV/VIS Spectroscopy 
 
 9.0 Example Chromatography 
 9.1 Blank 
 
 Blank 
 
 120- 
 100- 
 
 20- 642 
 1.9749 197 
 -20- 
 a ' t ' ' 7 Li bj 
 7 8 9 10 14 12 13 14 15 
 Time [min] 
 = ooON dt =or nonc + 
 Oo 
 9.2. Working Standard 
 18AS026 0.1mg/mi 
 120- 
4.141 100- aspberry ketone

 80- 
 
 0 e242 40 
 —a 
 
 0 { 2 3 4 5 6 7 8 
 Time [min] 
 den _ — 
 10 14 12 13 14 15 
 +OC 
 9.3. Sample 
 210138 0.1mg/ml 
 16> 
 144 
4.135 aspberry ketone

 UAm 
 
 ~ « 4~ 4 4~ do ~ 4 5 6 7b] 8t 9 10 3011 12 13. «14 15
 Time {min] 
 4 fo 
 
 

[SOP 

 Standard Operating Procedure SON _ 
 Raspberry Ketone and Zi° ngerone Determi°n ati° on by O ev Page 8 of 8 
 : D-727 6 
 HPLC using UV/VIS Spectroscopy 
 
 10.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 3 | 11/18/13 | calculation for standard preparation, added dissolution buffer description, error correction | 13-1055 | B. Johns |
| 4 | 04/01/16 | Biennial review: Updated SOP to current format. | 16-0203 | J. Maignan |
| 5 | 01/03/19 | Updated SOP. Added zingerone based on validation. Update for consistency with current methods, add recommended | 19-0007 | J. Maignan |
| 6 | 06/01/22 | sequence section, replace requirements with system suitability CC- section, add example chromatography. | 22-0251 | S. Sassman |