Determination of Gingerols, Gingerdiones, and Shogaols by HPLC-UV

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the determination of gingerols,
 
 gingerdiones, and shogaols in raw materials and finished products by HPLC-UV.
 
 2.0 Scope 
 
 This procedure applies to the determination of gingerols, gingerdiones, and shogaols in raw
 
 materials and finished products in the QC Laboratory at Ion Labs. 
 
 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 ensure that this procedure is
 
 being followed. 
 
 3.3. It is the responsibility of QC Laboratory Management and/or Analytical Development to
 
 keep this procedure aligned with current practices. 
 
 4.0 Definitions 
 
 4.1 QC — Quality Control 
 
 4.2. HPLC-UV - High Performance Liquid Chromatography with Ultraviolet Detection
 
 5.0 References 
 
 5.1 PRTCL-20-0098, Protocol, Validation of an Analytical Method for the Determination of
 
 Gingerols by HPLC-UV 
 
 5.2 D-793, SOP, Cryogenic Grinding of Chewable Gels 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Gingerols, Gingerdiones, and D-732 ! : tea 
 Shogaols by HPLC-UV 
 
 6.0 Supplies 
 
 6.1 Chemicals 
 
 6.1.1 Capsaicin Reference Standard 
 
 6.1.2 USP Ginger Constituent Mixture Reference Standard or equivalent
 
 6.1.3. Acetonitrile (HPLC grade) 
 
 6.1.4 Methanol (HPLC grade) 
 
 6.1.5 Phosphoric acid (ACS reagent or HPLC grade) 
 
 6.1.6 Water (>18 MQecm) 
 
 6.2 Glassware 
 
 6.2.1 Volumetric glassware as required for standard and sample preparations
 
 6.2.2 Luer-lock syringe and 0.45um filter 
 
 6.2.3 Adjustable pipette and tips 
 
 6.2.4 1.5-mL or 2-mL Eppendorf centrifuge tubes 
 
 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 Wrist action shaker 
 
 6.3.4 Centrifuge 
 
 7.0 Mobile Phase 
 
 7.1. Mobile Phase A 
 7.1.1 Transfer 550 mL of acetonitrile to a 1-L media bottle. 
 
 7.1.2 Add 10.0 mL of methanol. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Page 
 Determination of Gingerols, Gingerdiones, and D-732 
 3 of 9 
 Shogaols by HPLC-UV 
 7.1.3 Add 440 mL of water. 
 
 7.1.4 Add 0.44 mL of phosphoric acid. 
 
 7.1.5 Mix well. 
 
 7.2. Mobile Phase B 
 
 7.2.1 Use acetonitrile. 
 
 7.3 Diluent 
 
 7.3.1. Use methanol. 
 
 8.0 Stock Standard 
 
 8.1 The concentration of capsaicin in the Stock Standard is 0.3 mg/mL.
 
 8.2 The purity of commercially available capsaicin reference standards may be much lower
 
 than 100%. Use the certified purity listed on the certificate of analysis to calculate the
 amount of reference standard required to generate a capsaicin concentration of 0.3
 
 mg/mL. 
 
 8.3 Accurately weigh and transfer a quantity of capsaicin reference standard containing about
 30 mg of capsaicin to a 100-mL volumetric flask. 
 
 8.4 Dissolve and dilute to volume with Diluent. 
 
 9.0 Working Standard 
 
 9.1 Transfer 5.0 mL of the Stock Standard to a 25-mL volumetric flask.
 
 9.2 Dilute to volume with Diluent. 
 
 9.3. Alternatively, dilution may be performed directly into an HPLC vial using an adjustable
 
 pipet. 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Gingerols, Gingerdiones, and D-732 I : aah 
 Shogaols by HPLC-UV ‘ 
 
 10.0 System Suitability Solution 
 
 10.1 The System Suitability Solution may be stored at 2°C - 8°C for up to one year if the
 chromatogram obtained from the solution is similar in appearance to that provided with
 
 the CoA for USP Ginger Constituent Mixture Reference Standard. 
 
 10.2 Transfer the contents of one vial of USP Ginger Constituent Mixture Reference Standard
 
 to a 2-mL HPLC vial. 
 
 10.3 Add 1.0 mL of Working Standard. 
 
 10.4 Mix by vortex to dissolve. 
 
 11.0 Sample Solution 
 
 11.1 Specific sample testing details are provided in each products profile. If a specific testing
 
 details section is not available, then follow preparation procedure as described below.
 
 11.2 For raw materials: weigh no less than 20 mg into a suitably sized volumetric flask of no
 less than 25 mL volume to generate a total gingerols concentration of about 0.35 mg/mL,
 
 add Diluent to 2/3 flask capacity, shake mechanically for 20 min, and dilute to volume
 
 using Diluent. 
 
 11.3 For solid and liquid dose finished products: Combine and homogenize no less than ten
 dosage units. Based on the label claim and fill weight, serving size or tablet weight per
 
 dose, weigh no less than 50 mg of the pooled dosages into a suitably sized volumetric
 flask of no less than 25 mL to generate a total gingerols concentration of about 0.35
 
 mg/mL, add Diluent to 2/3 flask capacity, shake mechanically for 20 min, and dilute to
 
 volume using Diluent. 
 
 11.4 For chewable gels (gummies), homogenize at least 10 dosage units according to the
 procedure outlined in D-793 Cryogenic Grinding of Chewable Gels. Quickly weigh a
 
 portion of the pooled and homogenized dosages into a beaker. Use several small portions
 of Diluent to completely transfer the sample into a suitably sized volumetric flask to
 
 generate a total gingerols concentration of about 0.35 mg/mL, add Diluent to 2/3 flask
 capacity, shake mechanically for 20 min, and dilute to volume using Diluent.
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 
 Determination of Gingerols, Gingerdiones, and D-732 1 Page 
 5 of 9 
 Shogaols by HPLC-UV 
 1L.5 To manage large volumes, the sample can be initially dissolved in a smaller volume and
 
 a portion further diluted using Diluent to bring the total gingerols concentration to 0.35
 
 mg/mL. 
 
 11.6 Filter through a 0.45 um nylon syringe filter, discarding the first 2-3 mL before collecting
 a portion of the filtrate in an HPLC vial. Alternatively, the sample can be centrifuged at
 
 10,000 rpm for 5 min to remove particulates prior to analysis. 
 
 12.0 HPLC Parameters 
 
 12.1 Column: Acclaim 120 C18, 5um, 4.6 mm x 250 mm, or equivalent 
 
 12.2 Column Temperature: 30 °C 
 
 12.3 Flow rate: 1.0 mL/min 
 
 12.4 Wavelength: 282 nm 
 
 12.5 Injection Volume: 15 pL 
 
 12.6 Run Time: 94 minutes (samples, blank, and system suitability solution)
 
 12.7 Run Time: 15 minutes (working standard) 
 
 12.8 Gradient 
 
 Time (min) % A %B 
 0 100 0 
 65 100 0 
 67 0 100 
 77 0 100 
 79 100 0 
 94 100 0 
 
 12.9 Recommended Spectral Range (for Identification)- 200 nm to 400 nm 
 
 13.0 Recommended Sequence 
 
 13.1 Make two injections of Diluent 
 
 13.2 Make one injection of System Suitability Solution 
 
 13.3 Make five injections of Working Standard 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Page 
 Determination of Gingerols, Gingerdiones, and D-732 
 6 of 9 
 Shogaols by HPLC-UV 
 13.4 Make a single injection of each Sample Preparation 
 13.5 Make a single injection of Working Standard after every six samples and at the end of
 
 the run. 
 
 14.0 System Suitability Requirements 
 
 14.1 No significant (>0.5%) interfering peaks are present in the blank (Diluent) injection.
 
 14.2 The %RSD of the peak area in five consecutive injections of the Working Standard is
 NMT 2.5%. 
 
 14.3 The %RSD of the peak area in all injections of the Working Standard is NMT 3%.
 
 14.4 The USP resolution between 6-gingerol and capsaicin in the injection of System
 
 Suitability Solution is NLT 3.0. 
 
 14.5 The USP resolution between capsaicin and 6-shogaol in the injection of System Suitability
 Solution is NLT 10.0. 
 
 14.6 The tailing factors for 6-gingerol, capsaicin, and 6-shogaol in the injection of System
 Suitability Solution is NMT 2.0. 
 
 15.0 Retention Times 
 
 13,1 6-Gingerol =7.3 min 
 
 15.2 Capsaicin = 9.2 min (do not include capsaicin in the total) 
 
 13.3 8-Gingerol A = 15.0 min 
 
 15.4 6-Shogaol = 18.3 min 
 
 15.5 8-Gingerol B = 21.5 min 
 
 15.6 6-Gingerdiol = 25.1 min 
 
 15:7 6-Gingerdione = 26.7 min 
 
 15.8 10-Gingerol = 36.1 min 
 
 15.9 8-Shogaol = 38.9 min 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Page 
 Determination of Gingerols, Gingerdiones, and D-732 
 7 of 9 
 Shogaols by HPLC-UV 
 15.10 8-Gingerdione = 44.8 min 
 
 15.11 10-Shogaol = 53.7 min 
 
 16.0 Example Chromatograms 
 
 16.1 Working Standard 
 
 WSTD 19AS084 
 507 
 40+ 
 307 
 20; 
 104 
 UAm 
617.8 niciaspaC

912.9 1 o0 60 4 Be eg 14 «15

 Time [min] 
 16.2 System Suitability Solution 
 SYS SUIT R09440 
 507 
 40+ 
 305 
 20; 
 104 
 UAm 
666.6 !oregniG-6

862.7 niciaspaC

822.9 825

 oag 
 54 
 9 
220.91 5

 wD 
 foe] 
 ee 
 o = 434.2 782.4 
 | 
 037.8 
 505.21 
 > 
 71 
023.32 0472 3 4 5 67 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

 Time [min] 
 16.3. Raw Material Sample 
 R34442-1 
 40) 
 2 
 é 20; 
 oregniG-6 
672.7 & loregnic

 »= 
820.51 oagohS-6

353.81 loidregniG-6

209.42 loregniG-01

101.63 04

 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
 Time [min] 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Determination of Gingerols, Gingerdiones, and D-732 1 ‘ eG 
 
 Shogaols by HPLC-UV 
 
 16.4 Finished Product Sample 
 
 820548-1 
 2 = Se 6 re) : 
 
 a ai q e ° g s £ 8 2 5 5 : £ S tS S = e 
 oog ue goos i 1a 8 eee £3 
 
 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48 50
 Time [min] 
 
 17.0 Example Calculations 
 
 Note: Consult the raw material or finished product specification to see if shogaols should be
 included in the total gingerols. Gingerdiones are always included in the total.
 
 =! S Wtstq X P Se Adstock ¥ Vspt _ SS 
 Assay (%) = x — x 100 
 Ss Vstock Veta Splwt LA 
 Re Peak area for each gingerol present in the injection of Sample Solution
 Rg Mean Working Standard peak area (5 injections) 
 
 Wtsta Weight of reference standard used to prepare the Stock Standard (mg)
 
 P Purity of reference standard (% w/w) 
 
 Vstock Volume of the Stock Standard (mL) 
 AQstock Aliquot of the Stock Standard used to prepare the Working Standard (mL)
 
 Vea Volume of the Working Standard (mL) 
 
 Vspl Volume of Sample Solution (mL) 
 Splwt Sample weight (mg) 
 
 SS Serving size: Average weight of ten dosage units in mg for tablets and capsules,
 weight of a single serving from the theoretical formula in mg for liquids, or 1
 
 for raw materials 
 LA Label amount in mg (use 1 for raw materials) 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev ‘ 
 Determi°n ati° on of Gi°n gerols, Gi.n gerdi.o nes, and D-732 1 9 arf 
 Shogaols by HPLC-UV 
 
 18.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 11/16/20 | New N/A S. Sassman Added reference to D-793 for gummy sample prep. Added instruction to refer to test details for product specific sample | - | - |
| 1 | 05/17/23 | preparation. Added specific sample preparation instruction for CC- different dosage forms Added requirement for bracketing standard. Changed logo. | 23-0249 | S. Sassman |