Nobiletin Determination by HPLC using UV-Vis Spectroscopy

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1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 
 of nobiletin in raw materials and finished product dietary supplements using HPLC and UV/VIS
 spectrophotometry. 
 
 2.0 Scope 
 
 This procedure applies to the quantification and identification of nobiletin in raw materials and
 
 finished products. Nobiletin is a good chromophore and was measured at 280, 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 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 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2 | KH2PO4— Monobasic Potassium Phosphate 
 
 4.3. | H3PQ4— Phosphoric Acid 
 
 4.4 ACN — Acetonitrile 
 
 4.5 HeO- Water (= 18.2MQ-cm, 0.22um filtered) 
 
 46 Nobiletin —2-(3,4-Dimethoxypheny]l)-5,6,7,8-tetramethoxychromen-4-one
 
 4.7 QC-— Quality Control 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Nobiletin Determination by HPLC using UV/VIS D-763 1 : ie 
 
 Spectroscopy i 
 
 5.0 References 
 
 5.1 USP41-NF36 
 DZ Analysis of Synephrine in the Peel of Citrus Fruit, Immature Citrus Fruit and Decoctions
 
 of Chinese Medicinal Prescriptions Containing these Crude Drugs by Capillary
 
 Electrophoresis 
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are HPLC grade or better. 
 
 6.11 SEO 
 
 6.1.2 ACN 
 6.1.3 H3PQ, 
 
 6.1.4 KH2PO, 
 
 6.1.5 | Nobiletin reference standard 
 
 G.2 Glassware 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 6.2.2 Scintillation Vials 
 
 6.2.3 Mobile Phase Containers 
 
 6.2.4 Volumetric glassware as required by standard and sample preparations
 
 6.3 Disposables 
 6.3.1 10mL Pipette Tips 
 
 6.3.2 1mL Pipette Tips 
 
 6.3.3 200uL Pipette Tips 
 
 6.3.4 1.5mL centrifuge tubes 
 
 6.3.5 16mL Test Tubes 
 
 6.3.6 Disposable Plastic Luer Lock Syringe — 3mL, 6mL, or 10mL 
 
 6.3.7. Nylon Syringe Filters, 0.2um or 0.45pm 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Nobiletin Determination by HPLC using UV/VIS D-763 | . oes 
 Spectroscopy . 
 
 6.3.8 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting of a pump, autosampler, column oven
 
 and UV detector with a chromatographic data handling system 
 
 6.4.2 Analytical Balance 
 
 6.4.3 Ultrasonic bath 
 
 6.4.4 Vortex 
 
 6.4.5 Stir Plate 
 6.4.6 Eppendorf Centrifuge 
 
 6.4.7. Adjustable Pipette(s) 
 
 7.0 Preparation of Mobile Phase, Diluent, Samples, and Standards 
 
 7.1 Mobile Phase A — Acetonitrile 
 
 1s Mobile Phase B — 0.5g/L of KH2POs (aq) adjusted to pH 3.5 with H3PO4
 
 7.2.1. Prepared by adding 500mg KH2POs to a 1L volumetric flask and adding about
 
 950mL H20. Adjust pH to 3.5 with H3PO4. After pH is adjusted, dilute to
 volume with water. 
 
 7.3 Diluent—~ Mobile Phase A and Mobile Phase B (25:75) 
 
 7.4 Standard Preparation 
 
 7.4.1. Accurately weigh and transfer about 6.25 mg of reference standard into a 25-
 
 mL volumetric flask. 
 7.4.2 Dissolve in and dilute to volume using Diluent. 
 
 7.4.3 Standard preparation may be scaled up if necessary. 
 
 7.5 Sample Preparation 
 
 7.5.1. 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, maintaining concentration within the linear range listed below.
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Nobiletin Determination by HPLC using UV/VIS D-763 I rie 
 Spectroscopy 
 
 7.5.1. For raw materials: weigh no less than 25 mg into a suitably sized volumetric
 
 flask of no less than 25 mL. Dilute to volume using Diluent, and sonicate for 15
 
 minutes. 
 
 fake: For solid dose finished products: Combine and homogenize no less than ten
 dosage units. Based on the label claim and fill weight (for capsules) or tablet
 
 weight per dose, weigh no less than 25 mg of the pooled dosages into a suitably
 sized volumetric flask of no less than 25 mL, dilute to volume using Diluent,
 
 and sonicate for 15 minutes. 
 
 Td. For liquid dose finished products: Use a TC pipet to transfer no less than 2.0 mL
 of the product into a suitably sized volumetric flask of no less than 25 mL. Wipe
 
 the outside of the pipet, and rinse the pipet three times with Diluent collecting
 
 the rinses in the volumetric flask. Dilute to volume using Diluent.
 
 7.5.4 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 glass beaker.
 Dissolve the sample in Diluent, transfer the dissolved sample into a suitably
 
 sized volumetric flask of no less than 25 mL volume using small portions of
 Diluent to ensure complete transfer, and dilute to volume with Diluent.
 
 7.5.5 Perform further dilutions as required using Diluent. 
 
 7.5.6 Filter an aliquot of the sample solution through a 0.45 um membrane discarding
 
 the first 2 - 3 mL of filtrate before collecting a portion in a vial for analysis.
 
 7.5.6.1 Alternatively, the solution may be centrifuged to remove particulates
 provided that the final solution is clear. 
 
 8.0 Test Conditions 
 
 8.1 Gradient 
 
 Time YA %B 
 
 0.00 25 75 
 
 6.00 25 75 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Nobiletin Determination by HPLC using UV/VIS D-763 | : tie 
 
 Spectroscopy 
 
 12.0 90 10 
 
 15.0 90 10 
 
 15.1 a3 75 
 
 18.0 25 75 
 
 8.2 Column — Phenomenex Luna, C18 (2), Sum, 100A, LC column, 150mm x 4.6mm, or
 
 equivalent. 
 
 8.3 Flow Rate — 1.0mL/min 
 8.4 UV Detection — 280nm 
 
 8.5 Injection Volume — 10ynL 
 
 8.6 Column Temperature — 25°C 
 
 8.7 Recommended 3-D Spectral Range — 200nm to 400nm 
 
 8.8 Recommended Sequence 
 
 8.8.1 Make at least 2 injections of a Blank (Diluent). 
 
 8.8.2 Make five injections of the Working Standard. 
 
 8.8.3. Make a single injection of each Sample Preparation. 
 8.8.4 Makea single injection of the Working Standard after every ten samples and at
 
 the end of the run. 
 
 8.9 System Suitability 
 
 89.1 The %RSD of five consecutive injections of the Working Standard is NMT
 
 5.0%. 
 8.9.2 The %RSD of all standard injections is NMT 5%. 
 
 8.10 Recommended Column Wash and Storage 
 
 8.10.1 Rinse the column with H2O / ACN (90/10) at 1 mL/min for at least 15 min.
 
 8.10.2 Rinse the column with H2O / ACN (50/50) at 1 mL/min for at least 10 min.
 
 8.10.3 Store the column with H2O / ACN (50/50). 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Nobiletin Determination by HPLC using UV/VIS D-763 1 Mey 
 Spectroscopy 
 
 9.0 Calculations 
 
10.0 18-
 
 16- 
 14> 
 12- 
 UAm 
 9.1 Example calculations for determining finished product % label or raw material % purity
 R Wt aXP Vsnl SS 
 9.1.1 %9a ssay seen = —e x —SVetoa — x —saF xi—a x EON 
 Ry Sample peak area 
 Rg Mean standard peak area 
 Wtsiq Weight of reference standard in mg 
 Vista 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) 
 Vspi 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 
 Example Chromatography 
 &n 
 DO 
 &O 
 hcenroeameeL 
 netnaeemnneeeeeecL 
 10.1 Blank 
 Blank 
 ae) 
 S 
 S 
 D 
 os 
 1 2 3 4 5 6 7 8 9 10 11 #12 13 «14 #15 «160«©17~=«&W8 
 Time {min} 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Nobiletin Determination by HPLC using UV/VIS D-763 , : oe 
 0 
 Spectroscopy 
 10.2. Working Standard 
 
 21AS089 
 800- 3 2=& 
 700- 
 Be 
 600- 
 500+ 
 4 400- 
 300+ 
 200+ 
 co ts co 
 100+ | ges S 
 0 | ae x 
 3 4 5 6 7 8 9. 40 oT aa 48 ia es a i) 
 Time [min] 
 10.3 Sample 
 220457 
 S 
 300- iN 
 250- 
 a 
 5 2 
 << — = 
 © 150- 
 1007 
 ; = FILLE? 1 
 2 3 4 5 6 7 8 9 10 11 12 13 14 #15 #16 17~©«©18 
 Time {min] 
 
 11.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New N/A J. Maignan Update for consistency with current laboratory practices, simplify standard preparation, add specific sample prep instructions for 03/20/23 various dosage forms, add recommended sequence, add system CC-23-0144 S. Sassman suitability section, add column wash and storage recommendation, add example chromatography. Update logo and format. | - | - |