Berberine and Dihydroberberine Determination by HPLC using UV-Vis Spectroscopy

Original Document

Scanned document (image-only PDF)

Extracted Text

Searchable text extracted from PDF

1.0 Purpose 
 
 The purpose of this procedure is to define the method for the quantitation and/or identification
 
 of berberine and/or dihydroberberine 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 berberine and/or
 
 dihydroberberine in raw materials and finished products. Berberine is a good chromophore and
 was measured at 340, other wavelengths can be used to maximize signal to noise.
 
 3.0 Responsibility 
 
 Bel 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 UV/VIS — Ultraviolet and Visible Electromagnetic Spectrums 
 
 4.2. ACN -— Acetonitrile 
 
 4.3 MeOH — Methanol 
 4.4 DMSO - Dimethylsulfoxide 
 
 4.5 H2O2— Hydrogen Peroxide, 30% 
 
 4.6 CofA — Certificate of Analysis 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev : 
 Berberine and Dihydroberberine Determination by D-765 2 age 
 2 of 10 
 HPLC using UV/VIS Spectroscopy 
 
 4,7 H20 — Water 
 4.8 Berberine Chloride — 5,6-Dihydro-9, 10-dimethoxybenzo[g]-1,3-benzodioxolo[5,6-
 
 a]quinolizinium 
 
 4.9 Dihydroberberine — 9,10-Dimethoxy-6,8-dihydro-5H-[1,3]dioxolo[4,5-
 
 g]isoquinolino[3,2-a]isoquinoline 
 
 5.0 References 
 
 5.1 MV-LAB-18-178, Protocol, Berberine Determination using HPLC with UV/VIS
 Spectroscopy. 
 
 Died PRTCL-24-0030, Protocol, Validation of an Analytical Method for the Determination of
 
 Dihydroberberine by HPLC-UV 
 
 5.3 H. A. Weber , M. K. Zart , S. L. Ferguson , J. G. Greaves , A. P. Clark , R. K. Harris , D.
 Overstreet & C. Smith (2001) SEPARATION AND QUANTITATION OF 
 
 ISOQUINOLINE ALKALOIDS OCCURRING IN GOLDENSEAL, Journal of Liquid 
 Chromatography & Related Technologies, 24:1, 87-95, DOI: 10.1081/JLC-100000329
 
 6.0 Supplies 
 
 6.1 Chemicals: All reagents are ACS grade or better. 
 
 6.1.1 H20 @ 18.2 MQ:cm) 
 
 6.1.2 ACN 
 
 6.1.3 MeOH 
 
 6.1.4 DMSO 
 
 6.1.5 Ammonium acetate 
 
 6.1.6 H202 
 
 6.1.7. Berberine chloride reference standard 
 
 6.1.8 Dihydroberberine reference standard 
 6.2 Glassware (Use Red Glassware When Conducting Assay of Dihydroberberine)
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Berberine and Dihydroberberine Determination by D-765 Page 
 3 of 10 
 HPLC using UV/VIS Spectroscopy 
 
 6.2.1 HPLC vials, 12mm x 32mm with screw cap enclosures with septa
 
 6.2.2 Volumetric Pipets 
 6.2.3. Mobile Phase Containers 
 
 6.2.4 50mL Volumetric Flasks 
 
 6.2.5 100mL Volumetric Flasks 
 
 6.3. Disposables 
 
 6.3.1 10mL Pipette Tips 
 
 6.3.2 200uL, S00uL & 1000uL Pipette Tips 
 
 6.3.3 1.5mL microfuge tubes 
 
 6.3.4 Parafilm 
 
 6.3.5 Disposable Plastic Luer Lock Syringe —3mL, 6mL, or 10mL 
 
 6.3.6 Nylon or PTFE Syringe Filters, 0.45um 
 6.3.7 Weigh paper 
 
 6.4 Equipment 
 
 6.4.1 Suitable gradient HPLC system consisting ofa pump, autosampler, column oven
 
 and UV detector with a chromatographic data handling system 
 
 6.4.2 Analytical Balance 
 
 6.4.3. Micro Analytical Balance 
 
 6.4.4 Ultrasonic Bath 
 6.4.5 Heated Ultrasonic Bath (Required for Determination of Dihydroberberine.)
 
 6.4.6 Wrist Action Shaker 
 
 6.4.7 Stir Plate 
 
 6.4.8 Microfuge 
 
 6.4.9 10mL Pipette 
 
 6.4.10 ImL Pipette 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev : 
 
 Berberine and Dihydroberberine Determination by D-765 2 age 
 4 of 10 
 HPLC using UV/VIS Spectroscopy 
 6.4.11 200uL Pipette 
 
 7.0 Preparation of Mobile Phase, Diluent, Samples and Standards for 
 
 Determination of Berberine 
 
 Tal Mobile Phase A —- 10mM Ammonium Acetate in H20 
 
 7.1.1. Transfer ~770.8 mg of ammonium acetate to a 1000-mL mobile phase bottle.
 
 7.1.2 Add 1000 mL of H20, and mix to dissolve. 
 
 vee: Mobile Phase B — Acetonitrile 
 
 1a Diluent~ H20:ACN (50:50) 
 
 7.3.1 Transfer 500 mL of ACN to a 1000-mL mobile phase bottle. 
 
 7.3.2 Add 500 mL of H20, and mix well. 
 
 7.3.3 Equilibrate to room temperature prior to use. 
 
 7.4 Stock Standard Preparation (250 ug/mL) 
 
 7.4.1 Accurately weigh and transfer about 25 mg of reference standard into a 100-mL
 volumetric flask. 
 
 7.4.2 Dilute to volume using Diluent. 
 
 7.4.3 Sonicate for 15 minutes or until completely dissolved. 
 
 7.5 Working Standard Preparation (10 ug/mL) 
 
 7.5.1 Transfer 4.0 mL of the Stock Standard into a 100-mL volumetric flask.
 
 7.5.2 Dilute to volume using Diluent, and mix well. 
 
 7.6 Sample Preparation 
 7.6.1 Specific sample testing details are provided in each product profile. If a specific
 
 testing details section is not available, follow preparation procedure as described
 below, maintaining concentration within the linear range of this method.
 
 7.6.2 The linear range of the method is 0.01 mg/mL — 0.1 mg/mL. All working sample
 
 preparations must be within the linear range. 
 
 
 

[SOP 

 Standard Operating Procedure SOPNo | Rev 
 Berberine and Dihydroberberine Determination by | -765 2 peee 
 5 of 10 
 HPLC using UV/VIS Spectroscopy 
 7.6.3 For finished products, pool at least 20 dosage units as required and homogenize
 
 using a mortar and pestle. 
 
 7.6.4 Based on the label claim and fill or tablet weight for finished products or expected
 
 potency for raw materials, weigh an amount greater than the minimum weight of
 the analytical balance which is sufficient to generate a concentration within the
 
 linear range when dissolved in Diluent in an appropriately sized volumetric flask.
 To handle large volumes, the sample may be initially prepared at a higher
 
 concentration and subsequently diluted to within the linear range.
 
 1.65 Dilute to the final volume using Diluent, and sonicate for 15 minutes.
 
 7.6.6 Before injection, insoluble matter should be removed via filtration using a nylon
 syringe filter. Discard at least the first 0.5mL of filtrate before collecting a portion
 
 for analysis. Dilute filtrate as needed then add 1mL of the final dilution to an
 
 HPLC vial for analysis. 
 7.6.6.1 Alternatively, samples and standards can also be centrifuged at 6000
 
 RPM for 5 minutes in a microfuge to pellet insoluble matter.
 
 8.0 Preparation of Mobile Phase, Extraction Solvent, Diluent, Samples and 
 
 Standards for Determination of Dihydroberberine 
 
 8.1 Mobile Phase A — 10mM Ammonium Acetate in H2O 
 
 8.1.1 Transfer ~770.8 mg of ammonium acetate to a 1000-mL mobile phase bottle.
 
 8.1.2 Add 1000 mL of H20, and mix to dissolve. 
 
 8.2 Mobile Phase B — Acetonitrile 
 
 8.3. Extraction Solvent - MeOH:DMSO (50:50) 
 $32] Transfer 500 mL of MeOH to a 1000-mL mobile phase bottle. 
 
 8.3.2 Add 500 mL of DMSO, and mix well. 
 
 $.3,3 Equilibrate to room temperature prior to use. 
 
 8.4. Diluent- H»O:ACN (50:50) 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Berberine and Dihydroberberine Determination by D-765 2 Page 
 6 of 10 
 HPLC using UV/VIS Spectroscopy 
 
 8.4.1 Transfer 500 mL of ACN to a 1000-mL mobile phase bottle. 
 
 8.4.2 Add 500 mL of H20, and mix well. 
 8.4.3 Equilibrate to room temperature prior to use. 
 
 8.5. Dihydroberberine is extremely unstable in solution and quickly oxidizes to form dynamic
 
 mixtures of Berberine / Dihydroberberine. The present method overcomes this
 
 complication by quantitatively forcing this conversion (to Berberine) to completion.
 Both standard and sample preparations are incubated with hydrogen peroxide, enabling
 
 Dihydroberberine to be quantified as Berberine. 
 
 8.6 Stock Standard Preparation (100 ug/mL) (Use Red Glassware.) 
 8.6.1 Accurately weigh and transfer ~5 mg of reference standard into a 50-mL
 
 volumetric flask. 
 
 8.6.2 Add ~30-mL of Extraction Solvent and mechanically shake for 15 minutes.
 
 8.6.3 Fill to volume and sonicate at room temperature for 5 minutes.
 
 8.7. Working Standard Preparation (10 ug/mL) 
 
 8.7.1 Using a glass volumetric pipet, transfer 5.0 mL of the Stock Standard into a 50-
 mL volumetric flask then add 500-uL of H2O2. Swirl briefly to mix then seal the
 
 flask with parafilm. 
 
 8.7.2 Transfer the flask to a heated ultrasonicator (preheated to 40°C) and sonicate for
 1 hour. Remove from bath and let cool to room temperature. 
 
 8.7.3. Dilute to volume using Diluent, and mix well. 
 
 8.8 Sample Preparation (Use Red Glassware.) 
 
 8.8.1 Specific sample testing details are provided in each product profile. If a specific
 
 testing details section is not available, follow preparation procedure as described
 below, maintaining concentrations as prescribed below. 
 
 8.8.2 Pool at least 20 dosage units and homogenize as appropriate (e.g. grind tablets /
 capsule fill / powders / stick pack contents by mortar and pestle, cryogenically
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Page 
 Berberine and Dihydroberberine Determination by | 2-765 2 
 7 of 10 
 HPLC using UV/VIS Spectroscopy 
 powder and dissolve gummies, etc.) Transfer sufficient sample (based on the raw
 material manufacturer assay value / finished product profile) into a 50-mL
 
 volumetric flask in order to generate a sample stock that is ~0.1 mg/mL
 Dihydroberberine. (Do not deviate from this sample stock concentration.) Add
 
 ~ 30-mL Extraction Solvent and mechanically shake for 15 minutes then fill to
 
 volume and sonicate at room temperature for 5 minutes. 
 
 8.8.3 Using a glass volumetric pipet, transfer 5.0 mL of the stock sample into a 50-mL
 volumetric flask then add 500-uL of H2O2. Swirl briefly to mix then seal the flask
 
 with parafilm. Transfer the flask to a heated ultrasonicator (preheated to 40°C)
 and sonicate for 1 hour. Remove from bath and let cool to room temperature.
 
 Dilute to volume using Diluent and mix well. Filter a 5mL aliquot for analysis,
 discarding the first 3-4mL of filtrate. 
 
 9.0 Test Conditions 
 
 | Gradient 
 Time %A %B 
 
 0.00 70 30 
 
 8.00 20 80 
 
 8.01 70 30 
 
 12.00 70 30 
 
 Column — Phenomenex Kinetex, XB-C18, Sum, 100A, LC column, 250mm x 4.6mm, or
9.2 equivalent.

 9.3 Flow Rate — 1.0mL/min 
 
 9.4 UV Detection — 340nm 
 
 9.5 3D Spectral Range — 200nm — 500nm 
 
 9.6 Injection Volume - 10uL 
 
 97 Column Temperature — 25°C 
 
 
 

[SOP 

 Standard Operating Procedure SOP No Rev 
 Page 
 Berberine and Dihydroberberine Determination by D-765 
 8 of 10 
 HPLC using UV/VIS Spectroscopy 
 9.8 Retention Time — About 9.5 min 
 
 9.9 Recommended Sequence 
 9.9.1 Make at least 2 injections of a Blank (Diluent). 
 
 9.9.2 Make five injections of the Working Standard. 
 
 9.9.3 Make a single injection of each Sample Preparation. 
 
 9.9.4 Make a single injection of the Working Standard after every six samples and at
 
 the end of the run. 
 
 9.10 System Suitability 
 
 9.10.1 The %RSD of five consecutive injections of Working Standard is NMT 5.0%.
 9.10.2 The %RSD of all standard injections is NMT 5%. 
 
 9.11 Column Wash and Storage 
 
 9.11.1 Wash the column with ACN:H20 (50:50) at 1 mL/min for at least 15 min.
 
 9.11.2 Store the column with ACN:H20 (50:50). 
 
 10.0 Calculations 
 
 10.1 Example calculations for determining finished product % label or raw material % purity
 
 10.1.1 % assay= = “ = WterVq er,aX P x = V.A e SS x 100 
 
 Ry Sample peak area 
 
 Rg, Mean standard peak area 
 
 Wtstq Weight of reference standard in mg 
 Veta 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) 
 
 Vept Volume of the sample preparation accounting for dilutions in mL
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev . 
 Berberine and Dihydroberberine Determination by | 2-765 2 gcrio 
 HPLC using UV/VIS Spectroscopy 
 
 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 
 
 11.0 Example Chromatography and Spectrum 
 
 11.1. Working Standard 
 
 WSTD 
 gnire 
 250; 
 225; 
 2004 
 175+ 
 => 150+ 
 E1254 
 1004 
 75+ 
 50+ 
 25+ 
 05 115 225 3 35 4 45 5 55 6 65 7 75 8 85 9 95 10 105 11 11.5 12 
 Time [min] 
 
 11.2 Sample 
 210015 
 
 4007 
 350+ 
 3007 
 = 250+ 
 FS 2007 
 1507 
 100- 
 507 
 INR 
 05 1152 25 335 4 45 5 55 6 65 7 75 8 85 9 95 10 105 11 11.5 12 
 Time [min] 
 enirebreB 
 
 
 

[SOP 

 Standard Operating Procedure SOP No | Rev 
 Berberine and Dihydroberberine Determination by | 9-76 2 a ai 
 
 HPLC using UV/VIS Spectroscopy 
 
 11.3 UV Spectrum of Berberine 
 
 Berberine - 9.252 (20210507 170123.dx) 
 2007 fe ge sane Te, 
 100- a TM ee «es a a tlt nina 
 0-4 
 31007 
 2-200- 
 ~300-4 | 
 --540000--+ \ / 
 -600} \_ 
 490 200 210 220 230 240 250 260 270 280 290 300 310 320 330 340 350 360 370 380 390 400
 nm 
 
 12.0 Revision History 

| Rev | Date | Description of Changes | CCR # | By |
|-----|----------|------------------------|-------|----|
| 0 | 01/02/19 | New procedure. N/A J. Maignan Update for consistency with current methods and lab practices, add | - | - |
| 1 | 08/05/22 | recommended sequence, add system suitability section, add expected CC- retention time, add linear range, simplify standard preparation. 2 03/26/24 U A p d d d a e t d e d f e o t r e c r o m n i s n i a s t t i e o n n c o y f w D i i t h h y c d u r r o r b e e n r t b e m r e i t n h e o . d s and lab practices. CO er OUT Peay | 22-0334 | S. Sassman |